Serine protease molecules and therapies

ABSTRACT

Cell-targeted serine protease constructs are provided. Such constructs can be used in methods for targeted cell killing such as for treatment cell of proliferative diseases (e.g., cancer). In some aspects, recombinant serine proteases, such as Granzyme B polypeptides, are provided that exhibit improved stability and cell toxicity. Methods and compositions for treating lapatinib or trastuzumab-resistant cancers are also provided.

This application is a divisional of U.S. application Ser. No.14/322,485, filed Jul. 2, 2014, which is a divisional of U.S.application Ser. No. 14/046,211, filed Oct. 4, 2013, now U.S. Pat. No.9,096,840, which claims the benefit of U.S. Provisional Application Nos.61/709,763, filed Oct. 4, 2012; 61/762,173, filed Feb. 7, 2013; and61/762,216, filed Feb. 7, 2013, each of which is incorporated herein byreference in its entirety.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named“CLFRP0395USD3.txt”, which is 68 KB (as measured in Microsoft Windows®)and was created on Mar. 8, 2018, is filed herewith by electronicsubmission and is incorporated by reference herein.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates generally to the field of molecularbiology and recombinant protein production. More particularly, itconcerns modified serine protease polypeptides, such as granzymes, andcell-targeting constructs comprising such polypeptides.

2. Description of Related Art

The successful development of targeted therapeutics (e.g., for cancerapplications) depends on the identification of ligands and antigensspecific for target cells, generation of molecules capable of targetingthose components specifically and, finally, use of highly toxicmolecules for killing of target cells. Immunoconjugates composed ofantibodies and small, toxic drugs or radioisotopes have beensuccessfully tested in vitro, in animal models and have demonstratedactivity in the clinical setting. In addition to the use of smallmolecules for the toxin component, a number of highly cytotoxic proteincomponents, such as diphtheria toxin, ricin A-chain, Pseudomonasexotoxin, and gelonin (rGel), have been used for targeted therapies.However, problems such as capillary leak syndrome, immunogenicity andinadvertent toxicity (to non-targeted cells) continue to limitimplementation of successful therapy, especially for long-term orchronic applications. Thus, there remains a need for highly specific andhighly active toxin molecules and cell-targeting constructs comprisingsuch molecules.

SUMMARY OF THE INVENTION

Certain embodiments of the invention concern truncated serine proteasepolypeptides and fusion proteins comprising such serine proteases. Asused herein, a truncated serine protease polypeptide refers to anengineered serine protease that is truncated such that the leadersequence, positioned N-terminally relative to a IIGG, IVGG or ILGGsequence has been removed or replaced with a heterologous sequence.Examples of such truncated serine protease polypeptides are shown inFIG. 1. In some aspects, a truncated serine protease polypeptide isconjugated to, or fused with, a cell targeting moiety to provide acell-targeted cytotoxic construct. Such constructs can be used, forexample, in the treatment of cell proliferative diseases, such ascancer.

Thus, certain embodiments there is provided a recombinant polypeptidecomprising a cleavage site that is susceptible to cleavage by a selectedprotease fused to a truncated serine protease having an IIGG, IVGG orILGG at its N-terminus, such that, upon cleavage of the polypeptide bythe selected protease, the truncated serine protease having anN-terminal isoleucine will be released from the polypeptide. In someaspects, the protease cleavage site is a caspase, furin, granzyme B orfactor Xa cleavage sequence. In some aspects, the protease cleavage siteis for an intracellular or extracellular protease. For instance, thecleavage site can be a caspase 1-10 cleavage site (e.g., YEVD, WEHD,DVAD, DEHD, DEVD, DMQD, LEVD, LEHD, VEID, VEHD, IETD, LETD or IEAD), afurin cleavage site (RVRR), a granzyme B cleavage site (IEPD) or afactor Xa cleavage site ((I/A)(E/D)GR; SEQ ID NO: 28). Furthermore, inpreferred aspects, the recombinant polypeptide further comprises acell-binding moiety, positioned N-terminally relative to the cleavagesite. For example, the cell-binding moiety can be an antibody or aligand (e.g., VEGF or BLyS), such as a moiety that binds to GP240, 5T4,HER1, HER2, CD-33, CD-38, VEGFR-1, VEGFR-2, CEA, FGFR3, IGFBP2, Fn14 orIGF-1R.

In some specific aspects a truncated serine protease for use accordingto the embodiments comprises a sequence at least about 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical togranzyme B (SEQ ID NO: 1), granzyme A (SEQ ID NO: 46), granzyme H (SEQID NO: 47), granzyme K (SEQ ID NO: 48), granzyme M (SEQ ID NO: 49),Cathepsin G (SEQ ID NO: 50), Chymase (SEQ ID NO: 51), Myeloblastin (SEQID NO: 52), Kallikrein-14 (SEQ ID NO: 53), Complement factor D (SEQ IDNO: 54), PRSS3 protein (SEQ ID NO: 55), Trypsin-1 (SEQ ID NO: 56),Serine protease 57 (SEQ ID NO: 57) or PRSSL1 protein (SEQ ID NO: 58). Incertain aspects, the truncated serine protease is at least about 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto a human granzyme, such as granzyme B (GrB).

In yet further aspects of the embodiments a serine protease (e.g., GrBpolypeptide) or targeting agent of the embodiments further comprises anamino acid sequence comprising a Cys, wherein the amino acid sequence ispositioned C-terminally relative to the serine protease coding sequence.For example, the polypeptide can comprise the sequence SSCSGSA (SEQ IDNO: 12) positioned C-terminally relative to the serine protease codingsequence. In some aspects, the Cys (positioned C-terminally to serineprotease) can be used to conjugate the protease to a further moiety(e.g., a cell-targeting moiety), such as by forming a disulfide bridge.

In further embodiments the invention provides a recombinant Granzyme B(GrB) polypeptide having enhanced stability and/or activity. In someaspects, such GrB polypeptides can be conjugated or fused to acell-targeting moiety thereby providing a highly specific targetedcytotoxic construct. For example, the cell-targeting moiety can be acancer-cell targeting polypeptide (e.g., an antibody that binds to acancer cell-specific antigen). In such aspects, a method of targetedcancer therapy is provided that allows for specific killing of cancercells that express a given antigen while other cells are left intact. Inpreferred aspects, the GrB polypeptide and/or the targeting moiety arecomprised of a substantially human amino acid sequence. Thus, in someaspects, a polypeptide of the embodiments does not elicit a robustimmune response when administered to a human subject.

In certain specific aspects, a granzyme for use according to theembodiments is a GrB coding sequence comprising one or more amino aciddeletions and/or substitutions relative to a human GrB sequence such asSEQ ID NO: 1 (see also NCBI accession numbers nos. AAA75490.1 andEAW66003.1, incorporated herein by reference). For example, therecombinant GrB can be at least 80% identical to SEQ ID NO: 1 (e.g., atleast about or about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to SEQ ID NO: 1). In certain aspects, a GrB polypeptidecomprises one or more amino acid substitution to a corresponding aminoacid from a GrB of a different species. For instance, a substantiallyhuman GrB polypeptide can comprise 1, 2, 3, 4, 5, or more substitutionsat amino acid positions for a corresponding amino acid from one of theGrB polypeptides provided in FIG. 1 (e.g., a primate, porcine, bovine ormurine GrB). In some aspects, the recombinant GrB comprises one or moreof the following features: (a) an amino acid substitution or deletion atthe position corresponding to Asp 37; (b) an amino acid substitution ordeletion at the position corresponding to Asp 150; (c) an amino acidsubstitution or deletion at the position corresponding to Asn 51; (d) anamino acid substitution or deletion at the position corresponding to Asn84; and/or (e) an amino acid substitution or deletion at the positioncorresponding to Cys 210. In further aspects, a GrB polypeptidecomprises two, three, four or five of the features (a)-(e). In certainaspects, a recombinant GrB is defined as a substantially un-glycosylatedGrB polypeptide.

In a further embodiment a recombinant GrB polypeptide of the embodimentscomprises one or more of the following features: (a) an amino acidsubstitution or deletion at the position corresponding to Asp 37; (b) anamino acid substitution or deletion at the position corresponding to Asn51; (c) an amino acid substitution or deletion at the positioncorresponding to Asn 84; (d) an amino acid substitution or deletion atthe position corresponding to Arg 96; (e) an amino acid substitution ordeletion at the position corresponding to Arg 100; (f) an amino acidsubstitution or deletion at the position corresponding to Arg 102; (g)an amino acid substitution or deletion at the position corresponding toAsp 150; (h) an amino acid substitution or deletion at the positioncorresponding to Arg 201; (i) an amino acid substitution or deletion atthe position corresponding to Cys 210; (j) an amino acid substitution ordeletion at the position corresponding to Lys 221; (k) an amino acidsubstitution or deletion at the position corresponding to Lys 222; (l)an amino acid substitution or deletion at the position corresponding toLys 225; and/or (m) an amino acid substitution or deletion at theposition corresponding to Arg 226. Thus, in some aspects, a recombinantpolypeptide of the embodiments comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, or all 13 of the features (a)-(m).

In certain aspects, a recombinant GrB polypeptide lacks glycosylation atan amino acid position corresponding to human amino acid position Asn 51and/or Asn 84. In some aspects, a GrB polypeptide of the embodimentscomprises an amino acid substitution or deletion at a positioncorresponding to human amino acid position Asn 51 and/or Asn 84. Infurther aspects, a GrB polypeptide comprises a Arg, His, Lys, Asp, Glu,Ser, Thr, Gln, Cys, Gly, Pro, Ala, Val, Ile, Leu, Met, Phe, Tyr or Trpsubstitution at human amino acid position Asn 51 and/or Asn 84. Forexample, in one aspect, a recombinant GrB comprises an Ala, Ser, Thr,Lys or Gln substitution at a position corresponding to human amino acidposition Asn 51. Alternatively or additionally, a recombinant GrBcomprises an Ala, Ser, Thr, Arg or Gln substitution at a positioncorresponding to human amino acid position Asn 84.

In some aspects, a recombinant GrB polypeptide comprises an amino acidsubstitution or deletion at the positions corresponding to Lys 27 and/orArg 28. For example, a recombinant GrB may comprise a substitution atboth the positions corresponding to Lys 27 and Arg 28. In some cases,the substitution is selected from K27E or K27L and R28A. In stillfurther aspects, a recombinant GrB coding sequence one, two or threeamino acid substitutions or deletions at the positions corresponding to⁸²PKN⁸⁴. For example, in some specific aspects, a GrB coding sequencecomprises the sequence PVPN substituted at the positions correspondingto ⁸²PKN⁸⁴.

In further aspects, a recombinant GrB polypeptide of the embodimentscomprises an amino acid deletion or substitution (e.g., a substitutionof an amino acid having a polar side chain) at an amino acid positioncorresponding to human amino acid position Asp 37 and/or Asp 150. Thus,in some aspects a recombinant GrB polypeptide comprises a Arg, His, Lys,Glu, Ser, Thr, Asn, Gln, Cys, Gly, Pro, Ala, Val, Ile, Leu, Met, Phe,Tyr or Trp substitution at to human amino acid position Asp 37 and/orAsp 150. For example, a recombinant GrB can comprise a Ser, Thr, Gln,Glu or Asn substitution at a position corresponding to human amino acidposition Asp 37. Alternatively or additionally, a recombinant GrBcomprises a Ser, Thr, Gln, Glu or Asn substitution at a positioncorresponding to human amino acid position Asp 150.

In some aspects, a recombinant GrB polypeptide of the embodimentscomprises an amino acid substitution or deletion at a positioncorresponding to human amino acid position Arg 96, Arg 100, Arg 102, Arg201, and/or Arg 226. In further aspects, a GrB polypeptide comprises aAsn, His, Lys, Asp, Glu, Ser, Thr, Gln, Cys, Gly, Pro, Ala, Val, Ile,Leu, Met, Phe, Tyr or Trp substitution at human amino acid position Arg96, Arg 100, Arg 102, Arg 201, and/or Arg 226. In certain aspects, arecombinant GrB comprises a substitution at a position corresponding toArg 96, Arg 100, Arg 102, Arg 201, and/or Arg 226 for an amino acidresidue having a polar or positively charged side chain. For example, arecombinant GrB can comprise an Ala, Asn, Ser, Thr, Lys, His or Glnsubstitution at a position corresponding to human amino acid positionArg 96, Arg 100, Arg 102, Arg 201, and/or Arg 226. In still furtheraspects, a recombinant polypeptide comprises a deletion or substitutionat 2, 3, 4 or 5 of said Arg positions.

In certain aspects, a recombinant GrB polypeptide of the embodimentscomprises an amino acid substitution or deletion at a positioncorresponding to human amino acid position Lys 221, Lys 222 and/or Lys225. In further aspects, a GrB polypeptide comprises a Asn, His, Arg,Asp, Glu, Ser, Thr, Gln, Cys, Gly, Pro, Ala, Val, Ile, Leu, Met, Phe,Tyr or Trp substitution at human amino acid position Lys 221, Lys 222and/or Lys 225. In certain aspects, a recombinant GrB comprises asubstitution at a position corresponding to Lys 221, Lys 222 and/or Lys225 for an amino acid residue having a polar or positively charged sidechain. For example, a recombinant GrB can comprise an Ala, Asn, Ser,Thr, Arg, His or Gln substitution at a position corresponding to humanamino acid position Lys 221, Lys 222 and/or Lys 225. In still furtheraspects, a recombinant polypeptide comprises a deletion or substitutionat 2 or 3 of said Lys positions.

In still further aspects, a recombinant GrB polypeptide of theembodiments comprises an amino acid deletion or substitution at theposition corresponding to Cys 210. In some aspects, recombinant GrBcomprises a Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln, Gly, Pro, Ala,Val, Ile, Leu, Met, Phe, Tyr or Trp amino acid substitution at theposition corresponding to Cys 210. For example, the recombinant GrBpolypeptide can comprise an Ala, Val, Ile, Leu, Met, Ser, Thr, Asn, Phe,Tyr or Gln substitution at the position corresponding to Cys 210.

In yet further embodiments there is provided a composition comprising aplurality of recombinant GrB polypeptides (or fusion proteins orconjugates thereof) wherein at least about 90%, 95%, 98%, 99% or 99.5%of the GrB polypeptides have active enzymatic activity. In yet a furtherembodiment there is provided a composition comprising a plurality ofrecombinant GrB polypeptides (or fusion proteins or conjugates thereof)wherein at least about 90%, 95%, 98%, 99% or 99.5% of the GrBpolypeptides comprise an intact GrB coding sequence (i.e., a GrBpolypeptide sequence that has not been proteolytically cleaved). Instill yet a further embodiment there is provided a plurality ofrecombinant GrB polypeptides (or fusion proteins or conjugates thereof)wherein at least about 90%, 95%, 98%, 99% or 99.5% of the GrBpolypeptides are present as monomers (i.e., a single GrB polypeptide permolecule) in the composition. For example, any of the foregoingcompositions can be defined a pharmaceutical composition, such as anaqueous solution comprising the recombinant GrB polypeptides. In someaspects, a composition of the embodiments comprises a plurality ofrecombinant GrB polypeptides wherein at least about 90%, 95%, 98%, 99%or 99.5% of the polypeptides have (1) active enzymatic activity; (2)comprise an intact GrB amino acid sequence; and/or (3) are present inthe composition as a monomer relative to the GrB polypeptide.

In still a further embodiment there is provided a targeting agentcomprising (a) a truncated serine protease of the embodiments; (b) atargeting polypeptide; and (c) a cell penetrating peptide (CPP). Incertain aspects, the targeting polypeptide is a cancer cell-targetingpolypeptide, such as a polypeptide that binds to Her2/neu. For examplethe targeting peptide can comprise the scFv 4D5 sequence (SEQ ID NO:23). A CPP for use in a targeting agent of the embodiments may be any ofthe CPP sequences detailed herein. In a preferred aspect, the CPP is the“26” CPP, having the sequence of SEQ ID NO: 22. Thus, in some specificaspects, a targeting agent comprises from N-terminus to C-terminus (a) atruncated serine protease coding sequence (e.g., a granzyme); (i) afirst linker peptide; (b) a targeting polypeptide; (ii) a second linkerpeptide; and (c) a cell penetrating peptide (CPP) (e.g., having thesequence of SEQ ID NO: 22). A variety of linker peptides may be used inaccordance with the embodiments, for example, the first and/or secondlinker peptide can comprise the sequence of SEQ ID NO: 13. In still morespecific aspects, a targeting agent comprises from N-terminus toC-terminus (a) a recombinant GrB coding sequence (e.g., a wild typemammalian GrB coding sequence or a modified coding sequence of theembodiments) (i) a first linker peptide; (b) a targeting polypeptide;(ii) a second linker peptide; and (c) a cell penetrating peptide (CPP),having the sequence of SEQ ID NO: 22. Accordingly, in some aspects, atargeting agent comprises a polypeptide sequence at least about 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 24(e.g., a targeting agent comprising the sequence of SEQ ID NO: 24).

In yet a further embodiment there is provided a method of treating alapatinib or trastuzumab-resistant cancer (e.g., a breast cancer) in asubject comprising (a) identifying a subject having a lapatinib ortrastuzumab-resistant cancer; and (b) administering a Her2/neu-targetedtherapeutic to the subject, wherein the Her2/neu-targeted therapeutic islinked to a truncated serine protease of the embodiments (e.g., a GrBpolypeptide). For example, in some aspects, the subject has been, or iscurrently being treated with lapatinib or trastuzumab. In some preferredaspects, the Her2/neu-targeted therapeutic comprises a CPP sequence,such as the one of the targeting agents described supra. Thus, in someaspects, a composition is provided for use in treating a subject havinga lapatinib or trastuzumab-resistant cancer, the composition comprisingof a Her2/neu-targeted therapeutic comprising a truncated serineprotease of the embodiments.

In still a further embodiment a recombinant polypeptide is providedcomprising, from N-terminus to C-terminus, (a) a peptide comprising aprotease cleavage site; and (b) a truncated serine protease (e.g., arecombinant GrB polypeptide). In some aspects, a protease cleavage siteis positioned such that, upon cleavage by the protease, a serineprotease is produced having an isoleucine residue at its amino terminus(e.g., IIGG, IVGG or ILGG). Thus, in the case of GrB, upon proteasecleavage free GrB is released having an amino terminal sequence ofIIGGHEAK; SEQ ID NO: 27. In certain aspects, the protease cleavage siteis a site cleaved by a mammalian intracellular protease (e.g., aprotease that cleaves at the C-terminus of its recognition sequence).Examples of protease cleavage sites for use according to the embodimentsinclude, without limitation, a caspase 1-10 cleavage site (e.g., YEVD,WEHD, DVAD, DEHD, DEVD, DMQD, LEVD, LEHD, VEID, VEHD, IETD, LETD orIEAD), a furin cleavage site (RVRR), a granzyme B cleavage site (IEPD)or a factor Xa cleavage site ((I/A)(E/D)GR; SEQ ID NO: 28). In certainspecific aspects, a caspase-3 cleavage site is used and a recombinantpolypeptide of the embodiments comprises the caspase-3 cleavage sequenceof SEQ ID NO: 25. In still further aspects, a recombinant polypeptide ofthe embodiments is a GrB polypeptide and comprises the sequenceYVDEVDIIGGHEAK (SEQ ID NO: 26); RVRRIIGGHEAK (SEQ ID NO: 29);RVRRIIGGHEAK (SEQ ID NO: 30); (I/A)(E/D)GRIIGGHEAK (SEQ ID NO: 31);YEVDIIGGHEAK (SEQ ID NO: 32); WEHDIIGGHEAK (SEQ ID NO: 33); DVADIIGGHEAK(SEQ ID NO: 34); DEHDIIGGHEAK (SEQ ID NO: 35); DEVDIIGGHEAK (SEQ ID NO:36); DMQDIIGGHEAK (SEQ ID NO: 37); LEVDIIGGHEAK (SEQ ID NO: 38);LEHDIIGGHEAK (SEQ ID NO: 39); VEIDIIGGHEAK (SEQ ID NO: 40); VEHDIIGGHEAK(SEQ ID NO: 41); IETDIIGGHEAK (SEQ ID NO: 42); LETDIIGGHEAK (SEQ ID NO:43) or IEADIIGGHEAK (SEQ ID NO: 44). As detailed supra, in some aspects,a recombinant polypeptide may further comprise a cell penetratingpeptide (CPP) and/or a cell-binding moiety, such as a cell bindingmoiety positioned N-terminally relative to the protease cleavagesequence. In certain preferred aspects, the cell binding moiety is anantibody or an antigen-binding antibody fragment. Polynucleotidemolecules encoding recombinant polypeptides of the embodiments arelikewise provided.

In a specific embodiment there is provided a cell-targeting constructcomprising (a) a cell-binding moiety (e.g., an antibody orantigen-binding domain thereof); (b) a cleavage site that is susceptibleto cleavage by a selected protease; and (c) a GrB coding sequence (suchas one of the recombinant polypeptides provided herein) having an IIGGat its N-terminus, such that, upon cleavage of the polypeptide by theselected protease, the GrB having an N-terminal isoleucine will bereleased from the cell-targeting construct. As demonstrated herein suchcell-targeting construct are surprisingly stable even upon extendedexposure to serum and thereby provide ideal therapeutic agents.Accordingly, in some aspects, a method of providing a serum-stablecell-targeting construct is provided comprising obtaining acell-targeting construct comprising a GrB coding sequence positionedC-terminal relative to a heterologous protease cleavage site (e.g., acleavage site recognized by an intracellular protease).

In yet a further embodiment there is a provided a polynucleotidemolecule comprising a sequence that encodes a serine proteasepolypeptide or constructs of the embodiments. In some aspects, thepolynucleotide molecule is comprised in an expression cassette operablylinked to expression control sequences (e.g., a promoter, enhancer,intron, polyadenylation signal sequence or transcription terminatorsequence). In still further aspects, the polynucleotide molecule encodesa serine protease fusion protein such as cell-targeting construct of theembodiments.

In still a further embodiment a host cell is provided comprising anexpressible polynucleotide sequence encoding a truncated serine protease(or a cell-targeting construct) of the embodiments. In some aspects, thehost cell further comprises a truncated serine protease polypeptide ofthe embodiments. For example, a host cell of the embodiments can be amammalian cell (e.g., a cultured human cell), a yeast cell, a bacterialcell, a ciliate cell or an insect cell. Thus, in a further embodimentthere is provided a method of manufacturing a polypeptide comprising:(a) expressing a polynucleotide molecule encoding a truncated serineprotease of the embodiments in a cell under conditions to produce theencoded polypeptide; and (b) purifying the polypeptide from the cell.

In a further embodiment there is provided a truncated serine proteasepolypeptide of the embodiments, wherein the serine protease isconjugated to or fused with a cell-targeting moiety. For example, theserine protease polypeptide can be conjugated to a cell-targeting moietyby a thioester linkage (e.g., using a Cys residue comprised in theserine protease polypeptide or positioned C-terminally relative to theserine protease coding sequence). In some aspects, the cell-bindingmoiety is fused to a serine protease polypeptide to form a fusionprotein. In this aspect, a skilled artisan will recognize that thecell-targeting moiety should be positioned C-terminally relative to thetruncated serine protease coding sequence, thereby maintaining proteaseenzymatic activity. For example, in certain aspects, a cell-targetingmoiety can bind to a protein, carbohydrate or lipid expressed on a cell(e.g., specifically or preferentially expressed on a cancer cell).Examples of cell-targeting moieties are further detailed and exemplifiedbelow and include, without limitation, moieties that bind to GP240, 5T4,HER1, HER2, CD-33, CD-38, VEGFR-1, VEGFR-2, CEA, FGFR3, IGFBP2, IGF-1R,BAFF-R, TACI, APRIL, Fn14 or HER3.

In yet further aspects, a truncated serine protease or a cell-targetingconstruct is further conjugated to an imaging agent. For example, theimaging agent can be a radionuclide, a Mill contrast agent or anultrasound contrast agent. Thus, in some aspects, a method is providedfor imaging target cells in a subject comprising administering acell-targeting construct conjugated to an imaging agent to the subjectand imaging the target cells in the subject.

It will be understood that in certain cases, a fusion protein maycomprise additional amino acids positioned between the truncated serineprotease and the cell targeting polypeptide. In general these sequencesare interchangeably termed “linker sequences” or “linker regions.” Oneof skill in the art will recognize that linker regions may be one ormore amino acids in length and often comprise one or more glycineresidue(s) which confer flexibility to the linker. In some specificexamples, linkers for use in the current embodiments include, withoutlimitation, the 218 (GSTSGSGKPGSGEGSTKG; SEQ ID NO: 13), the HL (EAAAK;SEQ ID NO: 14) SSG and the G₄S (GGGGS; SEQ ID NO: 15) linkers. Suchlinker sequences can be repeated 1, 2, 3, 4, 5, 6, or more times orcombined with one or more different linkers to form an array of linkersequences. For instance, in some applications, a linker region maycomprise a protease cleavage site, such as the cleavage site recognizedby an endogenous intracellular protease. In this case when the celltargeting construct is internalized into a target cell proteolyticcleavage can separate the serine protease from a cell targeting moietyand/or other polypeptide domains. As such, cell targeting constructsaccording to this embodiment may have the advantage of enhancedintracellular activity of the targeted serine protease since potentialinterference from the cell targeting polypeptide will be reduced.

Cell targeting constructs according to the embodiments may compriseadditional amino acids attached to the serine protease, the celltargeting moiety, or both. For example, additional amino acids may beincluded to aid production or purification of a cell targetingconstruct. Some specific examples of amino acid sequences that may beattached to cell targeting moiety include, but are not limited to,purification tags (e.g., a T7, MBP. GST, HA, or polyHis tag),proteolytic cleavage sites, such as a thrombin or furin cleavage site,intracellular localization signals or secretion signals. Accordingly, incertain aspects, a cell-targeting construct of the embodiments comprisesa protease cleavage site (e.g., a furin cleavage site) positionedbetween a serine protease, such as GrB, and the cell-targeting moiety.

In still further aspects, a cell-targeting construct of the embodimentsfurther comprises a cell-penetrating peptide (CPP). As used herein theterms CPP and membrane translocation peptide (MTP) as usedinterchangeably to refer to peptide sequences that enhance the abilityof a protein to be internalized by a cell. Examples for CPPs for useaccording to the embodiments include, without limitation, peptidesegments derived from HIV Tat, herpes virus VP22, the DrosophilaAntennapedia homeobox gene product, protegrin I, as well as the T1 (SEQID NO: 19), T2 (SEQ ID NO: 20), INF7 (SEQ ID NO: 21) and 26 (SEQ ID NO:22) peptides exemplified herein. In certain aspects, a cell-targetingconstruct of the embodiments comprises CPP positioned between the serineprotease and the cell-targeting moiety or positioned C-terminallyrelative to the cell-targeting moiety. In certain aspects a CPP isseparated from a serine protease and/or a cell-targeting moiety by alinker sequence.

A cell targeting construct (e.g., comprising a cell-targeting moiety anda serine protease) according to the embodiments will desirably have twoproperties; (1) binding affinity for a specific population of cells and(2) the ability to be internalized into cells. It is envisioned,however, that even cell targeting constructs that are poorlyinternalized may be used in methods according to the embodiments.Methods well known to those in the art may be used to determine whethera particular cell targeting construct is internalized by target cells,for example by immunohistochemical staining or immunoblot ofintracellular extracts. It is also envisioned that, in certain cases,cell targeting moieties that cannot, by themselves, be internalized, maybe internalized in the context of the cell targeting constructsaccording to the embodiments. Cell targeting moieties for use in theembodiments include but are not limited to antibodies, growth factors,hormones, peptides, aptamers, avimers (see for example U.S. PatentPublns. 20060234299 and 20060223114, incorporated herein by reference)and cytokines. As discussed above, cell targeting moieties may beconjugated to a serine protease via a covalent or non-covalent linkage,and in certain cases the targeting construct may be a fusion protein.

In certain preferred aspects, cell targeting moieties for use in theembodiments are antibodies or fragments thereof. In general the termantibody includes, but is not limited to, polyclonal antibodies,monoclonal antibodies, single chain antibodies, humanized antibodies, adeimmunized antibodies, minibodies, dibodies, tribodies as well asantibody fragments, such as Fab′, Fab, F(ab′)₂, single domain antibody,Fv, or single chain Fv (scFv) antibody single domain antibodies, andantibody mimetics, such as anticalins, and any mixture thereof. In somecases the cell targeting moiety is a single chain antibody (scFv). In arelated aspect, the cell targeting domain may be an avimer polypeptide.Therefore, in certain cases, the cell targeting constructs of theembodiments are fusion proteins comprising a GrB polypeptide and a scFvor an avimer. For example, in some very specific aspects, the GrBpolypeptide is conjugated or fused to a 15A8, scFvMEL, ZME-018, scFv23,cetuximab or trastuzumab antibody. Likewise, a GrB polypeptide may befused or conjugated to and anti-CD-33 or anti-CD-38 antibody.

Thus, in some embodiments, the invention provides a cell targetingmoiety comprising a human antibody heavy chain and light chain, whereinthe antibody light chain, heavy chain or both comprise a truncatedserine protease of the embodiments positioned C-terminally relative tothe antibody light chain and/or heavy chain. For example, the antibodycan be a human IgG, such as an IgG1.

In still a further embodiment there is provided a cell-targetingconstruct comprising (a) a cell-targeting scFv antibody domain; (b) anantibody heavy chain constant (Fc) domain; and (c) a truncated serineprotease of the embodiments. For example, the cell-targeting constructcan comprise, from N- to C-terminus, (c) a truncated serine protease;(b) a Fc domain; and (a) a scFv domain. Alternatively, thecell-targeting construct can comprise, from N- to C-terminus, (a) a scFvdomain; (b) a Fc domain; (d) a peptide comprising a protease cleavagesite (e.g., cleavable by an intracellular protease) and (c) a truncatedserine protease of the embodiments. In some aspects, the cell-targetingconstruct can comprise additional elements, such a linkers or CPPs,fused the N-terminus, c-terminus or between any of the elements (a), (b)and/or (c). In certain specific aspects, the scFv of the cell-targetingconstruct binds to Fn14 and the serine protease is GrB, such as acell-targeting construct comprising the sequence of SEQ ID NO: 45 (orsequence at least about 85%, 90% or 95% identical to SEQ ID NO: 45).

In further aspects, a cell targeting moiety of the embodiments can be agrowth factor. For example, transforming growth factor, epidermal growthfactor, insulin-like growth factor, fibroblast growth factor, Blymphocyte stimulator (BLyS), heregulin, platelet-derived growth factor,vascular endothelial growth factor (VEGF), or hypoxia inducible factormay be used as a cell targeting moiety according to the embodiments.These growth factors enable the targeting of constructs to cells thatexpress the cognate growth factor receptors. For example, VEGF can beused to target cells that express VEGFR-2 and/or VEGFR-1. In stillfurther aspects, the cell targeting moiety may be a polypeptide BLyS(see U.S. Patent Publn. 20060171919, incorporated herein by reference).

In yet further aspects, a cell targeting moiety may be a hormone. Someexamples of hormones for use in the embodiments include, but are notlimited to, human chorionic gonadotropin, gonadotropin releasinghormone, an androgen, an estrogen, thyroid-stimulating hormone,follicle-stimulating hormone, luteinizing hormone, prolactin, growthhormone, adrenocorticotropic hormone, antidiuretic hormone, oxytocin,thyrotropin-releasing hormone, growth hormone releasing hormone,corticotropin-releasing hormone, somatostatin, dopamine, melatonin,thyroxine, calcitonin, parathyroid hormone, glucocorticoids,mineralocorticoids, adrenaline, noradrenaline, progesterone, insulin,glucagon, amylin, erythropoitin, calcitriol, calciferol,atrial-natriuretic peptide, gastrin, secretin, cholecystokinin,neuropeptide Y, ghrelin, PYY3-36, insulin-like growth factor-1, leptin,thrombopoietin or angiotensinogen. As discussed above targetingconstructs that comprise a hormone can be used in methods of targetingcell populations that comprise extracellular receptors for the indicatedhormone.

In yet still further aspects of the embodiments, cell targeting moietiesmay be cytokines. For example, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8,IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL-16, IL-17, IL-18, IL-19,IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29,IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, granulocyte-colonystimulating factor, macrophage-colony stimulating factor,granulocyte-macrophage colony stimulating factor, leukemia inhibitoryfactor, erythropoietin, granulocyte macrophage colony stimulatingfactor, oncostatin M, leukemia inhibitory factor, IFN-γ, IFN-α, IFN-β,LT-β, CD40 ligand, Fas ligand, CD27 ligand, CD30 ligand, 4-1BBL, TGF-β,IL 1α, IL-1β, IL-1RA, MIF, TNF-like weak inducer of apoptosis (TWEAK)and IGIF may all be used as targeting moieties according to theembodiments.

From the foregoing description it will be clear to one of skill in theart that cell targeting constructs according to the embodiments maytarget particular populations of cells depending on the cell targetingmoiety that is employed. For instance, the cell targeting moiety may bean infected cell targeting moiety. In this case, the cell targetingmoiety may bind to a cellular protein that is primarily expressed on thesurface of cells that are infected by a pathogen, such as bacteria, aprotozoan or a virus. In certain other aspects, the cell targetingmoiety may bind to a factor encoded by the pathogen, such as abacterial, protozoal or viral protein. In this aspect, it is envisionedthat cell targeting constructs may be indirectly targeted to cells bybinding to a pathogen before or as it enters a target cell. Thus, thetransit of a pathogen into a cell may, in some instances, mediateinternalization of the targeting construct. In additional aspects, celltargeting moieties may bind to polypeptides encoded by the pathogen thatare expressed on the surface of infected cells. For example, in the caseof a cell infected with human immunodeficiency virus (HIV), a celltargeting moiety may bind to, for example, gp120. It is envisioned thatany of the foregoing methods may be used to limit the spread ofinfection. For example, delivery of a serine protease (e.g., GrB) to theinfected cell may induce apoptosis or sensitize a cell to undergoapoptosis.

In some aspects of the embodiments a cell-targeting moiety can bedefined as an immune cell targeting moiety. In this case, the celltargeting moiety may bind to and/or be internalized by a cell surfacemolecule that is expressed on a specific populations of immune cells.Thus, targeting a serine protease to certain types of immune cells maybe used, for example, to treat autoimmune diseases or lymphomas.

In still further aspects of the embodiments a cell targeting moiety canbe a cancer cell targeting moiety. It is well known that certain typesof cancer cells aberrantly express surface molecules that are unique ascompared to surrounding tissue. Thus, cell targeting moieties that bindto these surface molecules enable the targeted delivery of serineproteases specifically to the cancers cells. For example, acell-targeting moiety may bind to and be internalized by a lung, breast,brain, prostate, spleen, pancreatic, cervical, ovarian, head and neck,esophageal, liver, skin, kidney, leukemia, bone, testicular, colon orbladder cancer cell. Thus, the effectiveness of a cancer cell-targetedserine protease may, in some cases, be contingent upon the expression orexpression level of a particular cancer marker on the cancer cell. Incertain aspects, there is provided a method for treating a cancerpatient with targeted serine protease comprising identifying whether (orto what extent) cancer cells of the patient expresses a particular cellsurface marker and administering a targeted serine protease therapy(optionally, in conjunction with a further anticancer therapy) to apatient identified to have a cancer expressing the particular cellsurface marker. In further aspects, the dose of a targeted serineprotease therapy can be adjusted depending on the expression level of acell surface marker on the cancer cells.

Accordingly, in certain embodiments, there is provided a method fortreating a cell proliferative disease comprising administering acell-targeting construct according to the embodiments. As used hereinthe phrase “cell proliferative condition” includes but is not limited toautoimmune diseases, cancers and precancerous conditions. For example,methods of the embodiments may be used for the treatment of cancers suchas lung, breast, brain, prostate, spleen, pancreatic, cervical, ovarian,head and neck, esophageal, liver, skin, kidney, leukemia, bone,testicular, colon, or bladder cancers. For example, there is provided amethod for treating a skin cancer, such as a melanoma, by administrationof a serine protease targeted to skin cancer cells. Likewise, there isprovided a method for treating a gp240 positive skin cancer comprisingadministering a serine protease of the embodiments that comprises ascFvMEL targeting moiety.

In some cases, cell-targeting constructs of the embodiments can used inconjunction with a further (e.g., a second) anticancer therapy. Thus, incertain instances, there is provided a method sensitizing cells to ananticancer therapy (e.g., a chemotherapy) by administering a celltargeting construct comprising a serine protease conjugated to a celltargeting moiety. In this case the cell targeting construct may beadministered prior to, concurrently with, or after administration of theanticancer therapy. For example, the anticancer therapy can be asurgical therapy, chemotherapy, radiation therapy, gene therapy orimmunotherapy. In some aspects, if the anticancer therapy is achemotherapy, in may be preferred that the chemotherapy comprise one ormore apoptosis inducing agents.

In yet further aspects of the embodiments there is provided a method fortreating an autoimmune disease or an inflammatory disease comprisingadministering a cell targeting construct according the embodiments. Forexample, cell targeted-serine protease may be used in the treatment ofrheumatoid arthritis, psoriasis, osteoarthritis, inflammatory boweldisease, type 1 diabetes, tissue or organ rejection or multiplesclerosis. In these aspects, cell targeting constructs may be used incombination with other treatment regimens, such as steroids.

Embodiments discussed in the context of a methods and/or composition ofthe invention may be employed with respect to any other method orcomposition described herein. Thus, an embodiment pertaining to onemethod or composition may be applied to other methods and compositionsof the invention as well.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising,” the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1A-C: Graphic alignments of various mammalian granzyme polypeptidesand serine proteases having high homology to granzymes. In each case thepolypeptide sequences provided are for the mature active polypeptide(i.e., lacking the N-terminal leader sequence). (A) Figure shows analignment of sequences for GrB from Homo sapiens (SEQ ID NO: 1; 100%);Pan troglodytes (SEQ ID NO: 2; 98%); Pan paniscus (SEQ ID NO: 3; 98%);Pongo abelii (SEQ ID NO: 4; 93%); Macaca nemestrina (SEQ ID NO: 5; 87%);Macaca mulatta (SEQ ID NO: 6; 87%); Macaca fascicularis (SEQ ID NO: 7;86%); Sus scrofa (SEQ ID NO: 8; 72%); Bos taurus (SEQ ID NO: 9; 72%);Rattus norvegicus (SEQ ID NO: 10; 70%); and Mus musculus (SEQ ID NO: 11;71%). Percent values in parenthesis indicate the percent identity tomature H. sapiens GrB. The amino acid positions corresponding to humanGrB Asp 37, Asn 51, Asn84, Asp150, and Cys210 are each indicated in boldand shaded. * next to H. sapiens indicates that certain sequence readsfor GrB indicate a “Q” at position 35 rather than the “R” depicted, seee.g., NCBI accession nos. AAA75490.1 versus EAW66003.1. (B) Figure showsan alignment of sequences for various mature Granzyme polypeptides fromHomo sapiens. Sequences are shown for granzyme B “Gzm B” (SEQ ID NO: 1),granzyme A “Gzm A” (SEQ ID NO: 46), granzyme H “Gzm H” (SEQ ID NO: 47),granzyme K “Gzm K” (SEQ ID NO: 49) and granzyme M “Gzm M” (SEQ ID NO:49). (C) Figure shows an alignment of sequences for serine proteasepolypeptides from Homo sapiens with high homology to granzymepolypeptides. Sequences are shown for mature granzyme B (SEQ ID NO: 1),Cathepsin G (SEQ ID NO: 50, NCBI accession no. P08311), Chymase (SEQ IDNO: 51, NCBI accession no. P23946), Myeloblastin (SEQ ID NO: 52, NCBIaccession no. P24158), Kallikrein-14 (SEQ ID NO: 53, NCBI accession no.Q9P0G3), Complement factor D (SEQ ID NO: 54, NCBI accession no. K7ERG9),PRSS3 protein (SEQ ID NO: 55, NCBI accession no. A1A508), Trypsin-1 (SEQID NO: 56, NCBI accession no. P07477), Serine protease 57 (SEQ ID NO:57, NCBI accession no. Q6UWY2) and PRSSL1 protein (SEQ ID NO: 58, NCBIaccession no. B7ZMF6). In the alignments “*” indicated identical aminoacid positions, “:” and “.” indicate highly similar or similar aminoacid positions respectively.

FIG. 2A-E: GrB polypeptides and constructs of the embodiments. A,Schematic showing general designs for GrB fusion constructs. Thepositions of membrane translocation peptides (MTP), endosomal cleavagepeptides (ECP), cytosolic cleavage peptides (CCP), and cell penetratingpeptides (CPP) are indicated. B, Schematic showing example GrBpolypeptides that can be used for chemical conjugation. Substitutions inGrB are indicated in each case (A, C210A; N1, D150N; d1; N51S; and d2N84A). For construct “CM”: “R to A” indicates substitutions R96A, R100A,and R102A; “R to K” indicates substitution R201K; and “K to A” indicatessubstitutions K221A, K222A, K225A, and R226A. C-E, Schematics showingthe designs of 50 GrB targeting constructs.

FIG. 3: Construction and testing of various fusion proteins comprisingVEGF₁₂₁ and GrB. Upper panel shows a schematic of four differentGrB-VEGF fusion proteins. GrB polypeptides are wild-type human sequence(WT), and in each case, GrB polypeptides are fused to VEGF₁₂₁ via a G₄Slinker sequence. The lower panel is a graph showing the GrB enzymaticactivity of each of the fusion proteins.

FIG. 4: Construction and testing of various fusion proteins comprisingGrB and ZME(VL-VH). Upper panel shows a schematic of four differentGrB-ZME fusion proteins. GrB polypeptides are wild-type human sequence(SL) or sequence with a substitution at N51S (d1/SL-1); N84A (d2/SL-2);or at both positions (d1,2/SL-3). In each case, GrB polypeptides arefused to ZME via a G₄S linker sequence. The lower panel is a graphshowing the GrB enzymatic activity of each of the fusion proteins thatwas expressed.

FIG. 5A-B: Construction and preparation of GrB-based fusion constructimmunotoxins. A, Schematic diagram of immunoGrB constructs containingscFv 4D5 and GrB without or with fusogenic peptide 26. B, PurifiedimmunoGrBs were analyzed by SDS-PAGE under reducing and non-reducingconditions.

FIG. 6A-D: Characterization and comparison of GrB-based fusions. A,K_(d) of immunoGrB constructs to Her2/neu ECD, Her2/neu-positive BT474M1 cells, and Her2/neu-negative Me180 cells by ELISA. B, Enzymaticactivity of GrB moiety of fusion proteins compared with native GrB. C,Internalization analysis of BT474 M1 cells and Me180 cells after 4 h oftreatment with 25 nM immunotoxin. Cells were subjected toimmunofluorescence staining with anti-GrB antibody (FITC-conjugatedsecondary), with PI nuclear counterstaining. D, Western blot analysis ofthe intracellular behavior of 25 nM immunoGrB in BT474 M1 cells.

FIG. 7A-C: Effects of immunoGrB on apoptotic pathways of BT474 M1parental, Herceptin resistant (HR), and Lapatinib resistant (LR) cells.A, Detection of apoptosis of GrB/4D5/26 by Annexin V/PI staining assay.Me180 cells served as a Her2/neu-negative control group. B, Western blotanalysis of cleavage and activation of caspases-3 and -9 as well as PARPby GrB-based fusion constructs. C, Western blot investigation ofapoptosis kinetics and specificity of GrB/4D5/26. Cells were treatedwith GrB/4D5/26 for up to 24 h with or without 100 μM zVAD-fmk for 24 hin parental or HR cells and for up to 48 h in LR cells.

FIG. 8A-B: Effects of immunoGrB on the mitochondrial pathway in BT474 M1parental, HR, and LR cells. A, Effects of GrB-based fusion proteins onthe upstream components Bcl-2 and BID in the mitochondrial pathway. B,Effects of immunoGrB on cytochrome c release and Bax translocation.

FIG. 9: Western blot analyses of the effects of GrB/4D5 and GrB/4D5/26in BT474 M1 parental, HR, and LR cells on Her2/neu and ER signalingpathways. Cells were treated with 100 nM immunoGrB for 24 or 48 h, andtotal cell lysates were quantified and further evaluated by western blotanalysis for pHer2/neu, pAkt, pmTOR, pERK, strogen receptor (ER),progesterone receptor m(PR), and PI-9 levels.

FIG. 10A-C: Tumor apoptotic activity of GrB/4D5/26 in BT474 M1 tumorxenografts. A, Mice with BT474 M1 flank tumors were intravenouslyinjected with saline or 44 mg/kg GrB/4D5/26 at the indicated times(arrows). Mean tumor volume was calculated as WxLxH as measured withdigital calipers. B, Immunofluorescence staining of tumor samples afteri.v. injection of saline and GrB/4D5/26. Twenty four hours afterinjection, animals were sacrificed and frozen tumor sections wereprepared and detected by anti-GrB antibody (green) and anti-mouse CD31antibody (red). Hoechst 33342 (blue) was used for DNA staining. C,Apoptosis detection in tumor tissue by TUNEL assay.

FIG. 11: Competitive cytotoxicity of GrB/4D5/26 with the addition ofHerceptin against MDA MB453 cells. MDA MB453 cells were plated into96-well plates and allowed to attach overnight. After that, the cellswere treated with different concentrations of GrB/4D5/26, or pretreatedwith 5 μM Herceptin for 6 h and then co-treated with variousconcentrations of GrB/4D5/26. After 72 h, the cells were stained withcrystal violet.

FIG. 12: Western blot analysis of the expression level of Her2/neu andPI-9 in a variety of cancer cells. Whole cell lysates (50 μg) wereanalyzed by SDS-PAGE and immunoblotted with anti-Her2/neu or anti-PI-9antibodies, followed by incubation with horseradish peroxidase-labeledsecondary antibodies and chemiluminescent detection. Actin was used asloading control.

FIG. 13: The effect of the endosomolytic reagent chloroquine on thecell-killing activity of GrB/4D5/26. BT474 M1 and the derivatives wereincubated with different concentrations of GrB/4D5/26 with or without 15μM chloroquine. After 72 h, the relative number of viable cells wasdetermined by crystal violet assay.

FIG. 14: Apoptotic effects of GrB/4D5 on Her2/neu positive and negativecells. To assess apoptosis, cells were seeded at 5×10⁵ cells per 6-wellplate, and then treated with 100 nM GrB/4D5 for 48 h. The development ofapoptotic cell death was detected by Annexin V/PI staining assay.

FIG. 15: Western blot characterization of BT474 M1 parental and itsderived Herceptin- and Lapatinib-resistant cells. The charactersincluded the expression and activation of Her- and ER-family members,the downstream ERK and Akt activity, and the endogenous GrB inhibitorPI-9 level in each cell line.

FIG. 16: mRNA level of PI-9 in BT474 M1 parental and resistant variants.Cells were harvested and RNA was extracted. The expression level of PI-9and β-actin mRNA was detected by semi-quantitative reverse-transcriptionPCR.

FIG. 17: Graph show the results of ELISA studies of the affinity of4D5-IgG1 vs. Herceptin® on the Her2 extra cellular domain (ECD).

FIG. 18: A schematic showing various GrB antibody fusion constructs.Upper left panel shows a basic IgG structure. Upper right panel shows anIgG structure comprising a GrB fused to the light chain via a cleavablelinker. Lower left panel shows an IgG structure comprising a GrB fusedto the heavy chain via a cleavable linker. Lower right panel shows GrBfused to a heavy chain (Fc) comprising single-chain antibodies fused toits C-terminus.

FIG. 19: The results of studies to measure the cytotoxicity of GrBconstructs fused at the N-terminus versus the C-terminus with andwithout a proteinase cleavage site to free active GrB. Lower panel showsa schematic of constructs “HCB” (HMEL scFv-G₄S-YVDEVD (SEQ ID NO:25)-GrB); “WH” (GrB-G₄S-INF7-HMEL scFv); and “HNB” (HMEL scFv-G₄S-GrB).Right panel, graph shows cytotoxicity of the constructs on MEF3.5−/−cells, which lack the HMEL scFv target receptor. Left panel, graph showscytotoxicity of the constructs on AAB527 cells which have the HMEL scFvtarget receptor.

FIG. 20: A reproduction of an SDS-PAGE gel used to separate antibodyfusions with wt GrB (right lanes) or GrB comprising the ‘A’ mutation(C210A). Results show that, in the case of antibody fusion with wt GrB,no defined band corresponding to the fusion protein was present. Incontrast, the GrB ‘A’ mutant produced a significant amount of intactfusion protein as evidenced by the defined band apparent in the gel.Migration positions for free antibody, fusion protein and a non-specificvector protein are indicated.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS I. The Present Invention

Recently, targeted cancer therapies have been developed for treatingvarious malignancies. By virtue of their cell targeting specificitythese agents can be both more effective and result in fewer side effectsas compared to conventional therapy, such as chemotherapy. However, evensuch targeted therapies often do not have a sufficiently high specificactivity to effectively kill a substantial proportion of the targetedcancer cells in patient. Likewise, even targeted therapies are often notsufficiently specific so as to avoid side effects that may result fromkilling of non-targeted cells in a patient. New therapeutics and methodsprovided herein address both of these deficiencies by providingcell-targeting constructs that are both highly toxic and highly specificto targeted cell populations.

As demonstrated herein the GrB “payload” polypeptides that are providedherein have both improved stability and activity. Each of the attributesresults in an increased toxicity of the GrB payload to targeted cells.Moreover because of the enhanced specific activity of the GrB molecules,lower dosages may be effective for therapy thereby reducing possibletoxic side effects of targeted therapies. In particular, recombinant GrBpolypeptides of the embodiments comprise one or more of the followingfeatures: (a) an amino acid substitution or deletion at the positioncorresponding to Asp 37; (b) an amino acid substitution or deletion atthe position corresponding to Asp 150; (c) an amino acid substitution ordeletion at the position corresponding to Asn 51; (d) an amino acidsubstitution or deletion at the position corresponding to Asn 84; and/or(e) an amino acid substitution or deletion at the position correspondingto Cys 210.

Thus, in some embodiments a recombinant Granzyme B (GrB) polypeptidehaving enhanced stability and activity is provided. In some aspects,such GrB polypeptides can be conjugated or fused to a cell-targetingmoiety, such as the 4D5 or ZME antibodies, thereby providing a highlyspecific targeted cytotoxic construct. In such aspects, a method oftargeted cancer therapy is provided that allows for specific targetedkilling of cancer cells that express a given antigen while other cellsare left intact. In preferred aspects, the GrB polypeptide and/or thetargeting moiety are comprised of substantially human amino acidsequence, which does not produce a robust immune response whenadministered to a human subject. For example, a cell-targeting constructof the embodiments can comprise from N-terminus to C-terminus arecombinant GrB polypeptide; optionally a linker; a CPP (such as T1 orINF7); and a cell-targeting moiety (such as ZME). Such a cell-targetingconstruct is exemplified in Examples 4, 5, and 7. In each case, theconstructs are shown to have highly specific and highly toxic activityrelative to target cells.

In a further aspect, a cell-targeting construct of the embodimentscomprises from N-terminus to C-terminus a serine protease polypeptide;optionally a linker; a cell-targeting moiety (such as 4D5); optionally asecond linker; and a CPP (such as CPP 26). Such constructs areexemplified herein in Example 8 and 11 and demonstrate highly selectivetoxicity to Her2-expressing cells. Interestingly, when these constructsincluded a CPP domain, not only was their cytotoxicity relative toHer2-expressing cells greatly increased, but they remained highlyeffective even against cells that had acquired resistance to anti-Her2therapies (see, e.g., the results shown in Table 12). Accordingly, thetargeting agents provided here are even effective against classes oftumors that have acquired resistance to other therapeutics that targetthe Her2 receptor. These new constructs can therefore be used to treatHer-2 positive cancers that have acquired resistance to therapy or toprevent resistance from being acquired in the first place by replacingcurrent therapeutics.

II. Serine Protease Polypeptides

As described in the foregoing summary, certain aspects of theembodiments concern a cell targeting constructs that comprises atruncated serine protease, such as one of the polypeptides shown inFIG. 1. In preferred aspects, a serine protease for use according to theembodiments is a human or substantially human polypeptide. For example,the truncated serine protease can be a granzyme selected from granzyme B(SEQ ID NO: 1), granzyme A (SEQ ID NO: 46), granzyme H (SEQ ID NO: 47),granzyme K (SEQ ID NO: 49) or granzyme M (SEQ ID NO: 49), or apolypeptide at least about 80%, 85%, 90% or 95% identical to one thesegranzyme polypeptides. In still further aspects, the serine protease isa protease from Homo sapiens having a N-terminal amino acid sequence ofIIGG, IVGG or ILGG (when in its mature, active form). For example, theserine protease can be Cathepsin G (SEQ ID NO: 50, NCBI accession no.P08311), Chymase (SEQ ID NO: 51, NCBI accession no. P23946),Myeloblastin (SEQ ID NO: 52, NCBI accession no. P24158), Kallikrein-14(SEQ ID NO: 53, NCBI accession no. Q9POG3), Complement factor D (SEQ IDNO: 54, NCBI accession no. K7ERG9), PRSS3 protein (SEQ ID NO: 55, NCBIaccession no. A1A508), Trypsin-1 (SEQ ID NO: 56, NCBI accession no.P07477), Serine protease 57 (SEQ ID NO: 57, NCBI accession no. Q6UWY2)or PRSSL1 protein (SEQ ID NO: 58, NCBI accession no. B7ZMF6) or apolypeptide at least about 80%, 85%, 90% or 95% identical to one theseprotease polypeptides.

In certain very specific aspects, a serine protease for use according tothe embodiments is a GrB polypeptide. Thus, one or more of the moleculesfor use in the current embodiments include, but are not limited to,human GrB (SEQ ID NO: 1) comprising one or more of the followingfeatures: (a) an amino acid substitution or deletion at the positioncorresponding to Asp 37; (b) an amino acid substitution or deletion atthe position corresponding to Asp 150; (c) an amino acid substitution ordeletion at the position corresponding to Asn 51; (d) an amino acidsubstitution or deletion at the position corresponding to Asn 84; and/or(e) an amino acid substitution or deletion at the position correspondingto Cys 210. For instance a GrB sequence for use according to the currentembodiments may comprise a GrB polypeptide that at least 70%, 80%, 90%,95%, 98% or more identical to human GrB. In certain aspects arecombinant GrB sequence is provided wherein one or more amino acid hasbeen substituted for an amino acid at a corresponding position of GrBfrom another species (other than human).

In certain cases, serine protease polypeptides or portions thereof maybe from a non-human source or may be from a homologous humanpolypeptide. For example, in the case of GrB, a polypeptide may compriseone or more amino acid substitutions to an amino acid at a correspondingposition in a Pan troglodytes (SEQ ID NO: 2); Pan paniscus (SEQ ID NO:3); Pongo abelii (SEQ ID NO: 4); Macaca nemestrina (SEQ ID NO: 5);Macaca mulatta (SEQ ID NO: 6); Macaca fascicularis (SEQ ID NO: 7); Susscrofa (SEQ ID NO: 8); Bos taurus (SEQ ID NO: 9); Rattus norvegicus (SEQID NO: 10); or Mus musculus (SEQ ID NO: 11) GrB (see, FIG. 1A).Likewise, a granzyme polypeptide of the embodiments may comprise one ormore amino acid substitutions to an amino acid at a correspondingposition in a different granzyme coding sequence (see, e.g., FIG. 1B).In yet further aspects, a truncated serine protease of the embodimentsmay comprise one or more amino acid substitutions to an amino acid at acorresponding position in a different, homologous, serine proteasecoding sequence (see, e.g., FIG. 1C). Because of the high homologyshared between these polypeptides, such substitutions for correspondingamino acid positions discussed above would be expected to result in acoding sequences that, when expressed, maintains protease activity.

In additional aspects, serine protease polypeptides may be furthermodified by one or more other amino substitutions while maintainingtheir enzymatic activity. For example, amino acid substitutions can bemade at one or more positions wherein the substitution is for an aminoacid having a similar hydrophilicity. The importance of the hydropathicamino acid index in conferring interactive biologic function on aprotein is generally understood in the art (Kyte and Doolittle, 1982).It is accepted that the relative hydropathic character of the amino acidcontributes to the secondary structure of the resultant protein, whichin turn defines the interaction of the protein with other molecules, forexample, enzymes, substrates, receptors, DNA, antibodies, antigens, andthe like. Thus such conservative substitution can be made in GrB andwill likely only have minor effects on their activity. As detailed inU.S. Pat. No. 4,554,101, the following hydrophilicity values have beenassigned to amino acid residues: arginine (+3.0); lysine (+3.0);aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine(+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline(−0.5±1); alanine (0.5); histidine −0.5); cysteine (−1.0); methionine(−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine(−2.3); phenylalanine (−2.5); tryptophan (−3.4). These values can beused as a guide and thus substitution of amino acids whosehydrophilicity values are within ±2 are preferred, those that are within±1 are particularly preferred, and those within ±0.5 are even moreparticularly preferred. Thus, any of the GrB polypeptides describedherein may be modified by the substitution of an amino acid, fordifferent, but homologous amino acid with a similar hydrophilicityvalue. Amino acids with hydrophilicities within +/−1.0, or +/−0.5 pointsare considered homologous. Furthermore, it is envisioned that serineprotease sequences may be modified by amino acid deletions,substitutions, additions or insertions while retaining its enzymaticactivity.

III. Cell Targeting Moieties

As discussed above cell targeting moieties according to the embodimentsmay be, for example, an antibody, a growth factor, a hormone, a peptide,an aptamer or a cytokine. For instance, a cell targeting moietyaccording the embodiments may bind to a skin cancer cell such as amelanoma cell. It has been demonstrated that the gp240 antigen isexpressed in a variety of melanomas but not in normal tissues. Thus, incertain aspects of the embodiments, there is provided a cell targetingconstruct comprising an GrB and a cell targeting moiety that binds togp240. In some instances, the gp240 binding molecule may be an antibody,such as the ZME-018 (225.28S) antibody or the 9.2.27 antibody. In aneven more preferred embodiment, the gp240 binding molecule may be asingle chain antibody such as the scFvMEL antibody. Therefore, in a veryspecific embodiment of the invention, there is provided a cell targetingconstruct comprising human GrB conjugated to scFvMEL.

In yet further specific embodiments of the invention, cell targetingconstructs may be directed to breast cancer cells. For example celltargeting moieties that bind to Her-2/neu, such as anti-Her-2/neuantibodies may conjugated to GrB. One example of such a cell targetingconstruct is a fusion protein comprising the single chain anti-Her-2/neuantibody scFv23 and GrB. Other scFv antibodies such as scFv(FRP5) thatbind to Her-2/neu may also be used in the compositions and methods ofthe current embodiments (von Minckwitz et al., 2005).

In certain additional embodiments, it is envisioned that cancer celltargeting moieties bind to multiple types of cancer cells. For example,the 8H9 monoclonal antibody and the single chain antibodies derivedtherefrom bind to a glycoprotein that is expressed on breast cancers,sarcomas and neuroblastomas (Onda et al., 2004). Another example are thecell targeting agents described in U.S. patent application no.2004005647 and in Winthrop et al., 2003 that bind to MUC-1, an antigenthat is expressed on a variety cancer types. Thus, it will be understoodthat in certain embodiments, cell targeting constructs according theembodiments may be targeted against a plurality of cancer or tumortypes.

Additionally, certain cell surface molecules are highly expressed intumor cells, including hormone receptors such as human chorionicgonadotropin receptor and gonadotropin releasing hormone receptor(Nechushtan et al., 1997). Therefore, the corresponding hormones may beused as the cell-specific targeting moieties in cancer therapy.

Since a large number of cell surface receptors have been identified inhematopoietic cells of various lineages, ligands or antibodies specificfor these receptors may be used as cell-specific targeting moieties. IL2may also be used as a cell-specific targeting moiety in a chimericprotein to target IL2R+ cells. Alternatively, other molecules such asB7-1, B7-2 and CD40 may be used to specifically target activated T cells(The Leucocyte Antigen Facts Book, 1993, Barclay et al. (eds.), AcademicPress). Furthermore, B cells express CD19, CD40 and IL4 receptor and maybe targeted by moieties that bind these receptors, such as CD40 ligand,IL4, IL5, IL6 and CD28. The elimination of immune cells such as T cellsand B cells is particularly useful in the treatment of autoimmunity,hypersensitivity, transplantation rejection responses and in thetreatment of lymphoid tumors. Examples of autoimmune diseases aremultiple sclerosis, rheumatoid arthritis, insulin-dependent diabetesmellitus, systemic lupus erythemotisis, scleroderma, and uviatis. Morespecifically, since myelin basic protein is known to be the major targetof immune cell attack in multiple sclerosis, this protein may be used asa cell-specific targeting moiety for the treatment of multiple sclerosis(WO 97/19179; Becker et al., 1997).

Other cytokines that may be used to target specific cell subsets includethe interleukins (IL 1 through IL15), granulocyte-colony stimulatingfactor, macrophage-colony stimulating factor, granulocyte-macrophagecolony stimulating factor, leukemia inhibitory factor, tumor necrosisfactor, transforming growth factor, epidermal growth factor,insulin-like growth factors, and/or fibroblast growth factor (Thompson(ed.), 1994, The Cytokine Handbook, Academic Press, San Diego). In someaspects, the targeting polypeptide is a cytokine that bind to the Fn14receptor, such as TWEAK (see, e.g., Winkles 2008; Zhou et al., 2011 andBurkly et al., 2007, incorporated herein by reference).

A skilled artisan recognizes that there are a variety of knowncytokines, including hematopoietins (four-helix bundles) (such as EPO(erythropoietin), IL-2 (T-cell growth factor), IL-3 (multicolony CSF),IL-4 (BCGF-1, BSF-1), IL-5 (BCGF-2), IL-6 IL-4 (IFN-β2, BSF-2, BCDF),IL-7, IL-8, IL-9, IL-11, IL-13 (P600), G-CSF, IL-15 (T-cell growthfactor), GM-CSF (granulocyte macrophage colony stimulating factor), OSM(OM, oncostatin M), and LIF (leukemia inhibitory factor)); interferons(such as IFN-γ, IFN-α, and IFN-β); immunoglobin superfamily (such asB7.1 (CD80), and B7.2 (B70, CD86)); TNF family (such as TNF-α(cachectin), TNF-β (lymphotoxin, LT, LT-α), LT-β, CD40 ligand (CD40L),Fas ligand (FasL), CD27 ligand (CD27L), CD30 ligand (CD30L), and4-1BBL)); and those unassigned to a particular family (such as TGF-β, IL1α, IL-1β, IL-1 RA, IL-10 (cytokine synthesis inhibitor F), IL-12 (NKcell stimulatory factor), MIF, IL-16, IL-17 (mCTLA-8), and/or IL-18(IGIF, interferon-γ inducing factor)). Furthermore, the Fc portion ofthe heavy chain of an antibody may be used to target Fcreceptor-expressing cells such as the use of the Fc portion of an IgEantibody to target mast cells and basophils.

Furthermore, in some aspects, the cell-targeting moiety may be a peptidesequence or a cyclic peptide. Examples, cell- and tissue-targetingpeptides that may be used according to the embodiments are provided, forinstance, in U.S. Pat. Nos. 6,232,287; 6,528,481; 7,452,964; 7,671,010;7,781,565; 8,507,445; and 8,450,278, each of which is incorporatedherein by reference.

Over the past few years, several monoclonal antibodies have beenapproved for therapeutic use and have achieved significant clinical andcommercial success. Much of the clinical utility of monoclonalantibodies results from the affinity and specificity with which theybind to their targets, as well as long circulating life due to theirrelatively large size. Monoclonal antibodies, however, are not wellsuited for use in indications where a short half-life is advantageous orwhere their large size inhibits them physically from reaching the areaof potential therapeutic activity.

Thus, in highly preferred embodiments, cell targeting moieties areantibodies or avimers. Antibodies and avimers can be generated tovirtually any cell surface marker thus, providing a method for targetedto delivery of GrB to virtually any cell population of interest. Methodsfor generating antibodies that may be used as cell targeting moietiesare detailed below. Methods for generating avimers that bind to a givencell surface marker are detailed in U.S. Patent Applns. 20060234299 and20060223114, each incorporated herein by reference.

Antibodies and Antibody-Like Targeting Moieties

As indicated above in some aspects the cell-targeting moiety is anantibody. As used herein, the term “antibody” is intended to includeimmunoglobulins and fragments thereof which are specifically reactive tothe designated protein or peptide, or fragments thereof. Suitableantibodies include, but are not limited to, human antibodies, primatizedantibodies, deimmunized antibodies, chimeric antibodies, bi-specificantibodies, humanized antibodies, conjugated antibodies (i.e.,antibodies conjugated or fused to other proteins, radiolabels,cytotoxins), Small Modular ImmunoPharmaceuticals (“SMIPs™”), singlechain antibodies, cameloid antibodies, antibody-like molecules (e.g.,anticalins), and antibody fragments. As used herein, the term“antibodies” also includes intact monoclonal antibodies, polyclonalantibodies, single domain antibodies (e.g., shark single domainantibodies (e.g., IgNAR or fragments thereof)), multispecific antibodies(e.g., bi-specific antibodies) formed from at least two intactantibodies, and antibody fragments so long as they exhibit the desiredbiological activity. In some aspects, the antibody can be a VHH (i.e.,an antigen-specific VHH) antibody that comprises only a heavy chain. Forexample, such antibody molecules can be derived from a llama or othercamelid antibody (e.g., a camelid IgG2 or IgG3, or a CDR-displayingframe from such camelid Ig) or from a shark antibody. Antibodypolypeptides for use herein may be of any type (e.g., IgG, IgM, IgA, IgDand IgE). Generally, IgG and/or IgM are preferred because they are themost common antibodies in the physiological situation and because theyare most easily made in a laboratory setting.

As used herein, an “antibody fragment” includes a portion of an intactantibody, such as, for example, the antigen-binding or variable regionof an antibody. Examples of antibody fragments include Fab, Fab′,F(ab′)2, Fc and Fv fragments; triabodies; tetrabodies; linearantibodies; single-chain antibody molecules; and multi specificantibodies formed from antibody fragments. The term “antibody fragment”also includes any synthetic or genetically engineered protein that actslike an antibody by binding to a specific antigen to form a complex. Forexample, antibody fragments include isolated fragments, “Fv” fragments,consisting of the variable regions of the heavy and light chains,recombinant single chain polypeptide molecules in which light and heavychain variable regions are connected by a peptide linker (“ScFvproteins”), and minimal recognition units consisting of the amino acidresidues that mimic the hypervariable region.

“Mini-antibodies” or “minibodies” are also contemplated for use with thepresent embodiments. Minibodies are sFv polypeptide chains which includeoligomerization domains at their C-termini, separated from the sFv by ahinge region (Pack et al., 1992). The oligomerization domain comprisesself-associating α-helices, e.g., leucine zippers, that can be furtherstabilized by additional disulfide bonds. The oligomerization domain isdesigned to be compatible with vectorial folding across a membrane, aprocess thought to facilitate in vivo folding of the polypeptide into afunctional binding protein. Generally, minibodies are produced usingrecombinant methods well known in the art. See, e.g., Pack et al.(1992); Cumber et al. (1992).

In some cases antibody-like molecules are protein scaffolds that can beused to display antibody CDR domains. The origin of such proteinscaffolds can be, but is not limited to, the structures selected among:fibronectin (see, e.g., U.S. Patent Publn. No. 20090253899, incorporatedherein by reference) and preferentially fibronectin type III domain 10,protein Z arising from domain B of protein A of Staphylococcus aureus,thioredoxin A or proteins with a repeated motif such as the “ankyrinrepeat” (Kohl et al., 2003), the “armadillo repeat”, the “leucine-richrepeat” and the “tetratricopeptide repeat.” The techniques for preparingand using various antibody-based constructs and fragments are well knownin the art. Additional antibody-like molecules, such as anti-calins aredescribed in detail in US Patent Publication Nos. 20100285564,20060058510, 20060088908, 20050106660, PCT Publication No. WO2006/056464and (Skerra, 2001), incorporated herein by reference.

Antibody-like binding peptidomimetics are also contemplated in thepresent embodiments. Liu et al. (2003) describe “antibody like bindingpeptidomimetics” (ABiPs), which are peptides that act as pared-downantibodies and have certain advantages of longer serum half-life as wellas less cumbersome synthesis methods. Likewise, in some aspects,antibody-like molecules are cyclic or bicyclic peptides. For example,methods for isolating antigen-binding bicyclic peptides (e.g., by phagedisplay) and for using such peptides are provided in U.S. Patent Publn.20100317547, incorporated herein by reference.

Monoclonal antibodies (MAbs) are recognized to have certain advantages,e.g., reproducibility and large-scale production. Embodiments of theinvention provide monoclonal antibodies of the human, murine, monkey,rat, hamster, rabbit and chicken origin. Due to the ease of preparationand ready availability of reagents, murine monoclonal antibodies willoften be preferred.

“Humanized” antibodies are also contemplated, as are chimeric antibodiesfrom mouse, rat, or other species, bearing human constant and/orvariable region domains, bispecific antibodies, recombinant andengineered antibodies and fragments thereof. As used herein, the term“humanized” immunoglobulin refers to an immunoglobulin comprising ahuman framework region and one or more CDR's from a non-human (usually amouse or rat) immunoglobulin. The non-human immunoglobulin providing theCDR's is called the “donor” and the human immunoglobulin providing theframework is called the “acceptor”. A “humanized antibody” is anantibody comprising a humanized light chain and a humanized heavy chainimmunoglobulin. Methods for humanizing antibodies such as those providedhere are well known in the art, see, e.g., Harvey et al., 2004,incorporated herein by reference.

IV. Fusion Proteins and Conjugates

A. Linkers

A variety of linkers can be used in truncated serine proteaseconstructsof the embodiments. In some aspects a linker can be a random string ofone or more amino acids (e.g., 2, 3, 4, 5, 10, 15, 20 or more aminoacids). Some specific linkers for use according the embodiments includethe 218 (GSTSGSGKPGSGEGSTKG; SEQ ID NO: 13), the HL (EAAAK; SEQ ID NO:14) and the G₄S (GGGGS; SEQ ID NO: 15) linkers (e.g., Robinson et al.,1998; Arai et al., 2004 and Whitlow et al., 1993, each incorporatedherein by reference).

In further aspects, a linker can serve as a way of separating differentdomains of a polypeptide construct, such as by proteolytic cleavage. Forexample, a linker region may comprise a protease cleavage site, such asthe cleavage site recognized by an endogenous intracellular protease. Instill further aspects, a protease cleavage site can be a site that isonly cleaved in certain cell types (e.g., a site cleaved by a viralprotease, such as HIV protease, which is only cleaved in infectedcells). Example of protease cleavage site for use according to theembodiments include, without limitation, thrombin, furin (Goyal et al.,2000) and caspase cleavage sites.

The cell targeting constructs of the embodiments may be joined by avariety of conjugations or linkages that have been previously describedin the art. In one example, a biologically-releasable bond, such as aselectively-cleavable linker or amino acid sequence may be used. Forinstance, peptide linkers that include a cleavage site for an enzymepreferentially located or active within a tumor environment arecontemplated. For example, linkers that are cleaved by urokinase,plasmin, thrombin, Factor IXa, Factor Xa, or a metalloproteinase, suchas collagenase, gelatinase, or stromelysin. In a preferred embodiment, alinker that is cleaved by an intracellular proteinase is preferred,since this will allow the targeting construct to be internalized intactinto targeted cells prior to cleavage.

Amino acids such as selectively-cleavable linkers, synthetic linkers, orother amino acid sequences such as the glycine rich linkers aredescribed above and may be used to separate proteinaceous components. Insome specific examples linkers for use in the current embodimentsinclude the 218 linker (GSTSGSGKPGSGQGSTKG) (SEQ ID NO: 13) or the G₄Slinker (GGGGS) (SEQ ID NO: 15). Additionally, while numerous types ofdisulfide-bond containing linkers are known that can successfully beemployed to conjugate the GrB with a cell targeting moiety, certainlinkers will generally be preferred over other linkers, based ondiffering pharmacologic characteristics and capabilities. For example,linkers that contain a disulfide bond that is sterically “hindered” areto be preferred, due to their greater stability in vivo, thus preventingrelease of the toxin moiety prior to binding at the site of action.

B. Conjugates

Additionally, any other linking/coupling agents and/or mechanisms knownto those of skill in the art can be used to combine the components ofthe present embodiments, such as, for example, antibody-antigeninteraction, avidin biotin linkages, amide linkages, ester linkages,thioester linkages, ether linkages, thioether linkages, phosphoesterlinkages, phosphoramide linkages, anhydride linkages, disulfidelinkages, ionic and hydrophobic interactions, bispecific antibodies andantibody fragments, or combinations thereof.

It is contemplated that a cross-linker having reasonable stability inblood will be employed. Numerous types of disulfide-bond containinglinkers are known that can be successfully employed to conjugatetargeting and therapeutic/preventative agents. Linkers that contain adisulfide bond that is sterically hindered may prove to give greaterstability in vivo, preventing release of the targeting peptide prior toreaching the site of action. These linkers are thus one group of linkingagents.

Another cross-linking reagent is SMPT, which is a bifunctionalcross-linker containing a disulfide bond that is “sterically hindered”by an adjacent benzene ring and methyl groups. It is believed thatsteric hindrance of the disulfide bond serves a function of protectingthe bond from attack by thiolate anions such as glutathione which can bepresent in tissues and blood, and thereby help in preventing decouplingof the conjugate prior to the delivery of the attached agent to thetarget site.

The SMPT cross-linking reagent, as with many other known cross-linkingreagents, lends the ability to cross-link functional groups such as theSH of cysteine or primary amines (e.g., the epsilon amino group oflysine). Another possible type of cross-linker includes thehetero-bifunctional photoreactive phenylazides containing a cleavabledisulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido)ethyl-1,3′-dithiopropionate. The N-hydroxy-succinimidyl group reactswith primary amino groups and the phenylazide (upon photolysis) reactsnon-selectively with any amino acid residue.

In addition to hindered cross-linkers, non-hindered linkers also can beemployed in accordance herewith. Other useful cross-linkers, notconsidered to contain or generate a protected disulfide, include SATA,SPDP and 2-iminothiolane (Thorpe et al., 1987). The use of suchcross-linkers is well understood in the art. Another embodiment involvesthe use of flexible linkers.

U.S. Pat. No. 4,680,338, describes bifunctional linkers useful forproducing conjugates of ligands with amine-containing polymers and/orproteins, especially for forming antibody conjugates with chelators,drugs, enzymes, detectable labels and the like. U.S. Pat. Nos. 5,141,648and 5,563,250 disclose cleavable conjugates containing a labile bondthat is cleavable under a variety of mild conditions.

U.S. Pat. No. 5,856,456 provides peptide linkers for use in connectingpolypeptide constituents to make fusion proteins, e.g., single chainantibodies. The linker is up to about 50 amino acids in length, containsat least one occurrence of a charged amino acid (preferably arginine orlysine) followed by a proline, and is characterized by greater stabilityand reduced aggregation. U.S. Pat. No. 5,880,270 disclosesaminooxy-containing linkers useful in a variety of immunodiagnostic andseparative techniques.

C. Cell Penetrating and Membrane Translocation Peptides

Furthermore, in certain aspects, library sequences can include segmentsof sequence that encode polypeptides having a known function, such as acell-binding domain or cell penetrating peptide (CPP) in the ORFsequence along with sequence derived from cDNA or randomized sequence(i.e., to generate an ORF encoding a fusion protein). Thus, in certainaspects, DNA molecules of the embodiments comprise an ORF that comprisesa CPP coding sequence along with a segment of library sequence (such asrandomized sequence), 5′ of the CPP coding sequence 3′ of the CPP codingsequence or both. As used herein the terms “cell penetrating peptide”and “membrane translocation domain” are used interchangeably and referto segments of polypeptide sequence that allow a polypeptide to crossthe cell membrane (e.g., the plasma membrane in the case a eukaryoticcell). Examples of CPP segments include, but are not limited to,segments derived from HIV Tat (e.g., GRKKRRQRRRPPQ; SEQ ID NO: 18),herpes virus VP22, the Drosophila Antennapedia homeobox gene product,protegrin I, Penetratin (RQIKIWFQNRRMKWKK; SEQ ID NO: 16) or melittin(GIGAVLKVLTTGLPALISWIKRKRQQ; SEQ ID NO: 17). In certain aspects the CPPcomprises the T1 (TKIESLKEHG; SEQ ID NO: 19), T2 (TQIENLKEKG; SEQ ID NO:20), 26 (AALEALAEALEALAEALEALAEAAAA; SEQ ID NO: 22) or INF7(GLFEAIEGFIENGWEGMIEGWYGCG; SEQ ID NO: 21) CPP sequence.

V. Administration and Pharmaceutical Formulations

In some embodiments, an effective amount of a cell targeting constructis administered to a cell. In other embodiments, a therapeuticallyeffective amount of the targeting construct is administered to anindividual for the treatment of disease. The term “effective amount” asused herein is defined as the amount of the cell targeted truncatedserine protease, such as GrB, of the present embodiments that isnecessary to result in a physiological change in the cell or tissue towhich it is administered either when administered alone or incombination with a cytotoxic therapy. The term “therapeuticallyeffective amount” as used herein is defined as the amount of thetargeting molecule of the present embodiments that eliminate, decrease,delay, or minimize adverse effects of a disease, such as cancer. Askilled artisan readily recognizes that, in many cases, cell targetedserine protease constructs may not provide a cure but may only providepartial benefit, such as alleviation or improvement of at least onesymptom. In some embodiments, a physiological change having some benefitis also considered therapeutically beneficial. Thus, in someembodiments, an amount of cell targeted serine protease (e.g., GrB) thatprovides a physiological change is considered an “effective amount” or a“therapeutically effective amount.” It will additionally be clear that atherapeutically effective amount may be dependent upon the inclusion ofadditional therapeutic regimens tat administered concurrently orsequentially. Thus it will be understood that in certain embodiments aphysical change may constitute an enhanced effectiveness of a secondtherapeutic treatment.

The cell targeting constructs of the embodiments may be administered toa subject per se or in the form of a pharmaceutical composition for thetreatment of cancer, autoimmunity, transplantation rejection,post-traumatic immune responses and infectious diseases, for example bytargeting viral antigens, such as gp120 of HIV. More specifically, thechimeric polypeptides may be useful in eliminating cells involved inimmune cell-mediated disorder, including lymphoma; autoimmunity,transplantation rejection, graft-versus-host disease, ischemia andstroke. Pharmaceutical compositions comprising the proteins of theembodiments may be manufactured by means of conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes. Pharmaceuticalcompositions may be formulated in conventional manner using one or morephysiologically acceptable carriers, diluents, excipients or auxiliarieswhich facilitate processing of the proteins into preparations which canbe used pharmaceutically. Proper formulation is dependent upon the routeof administration chosen.

In preferred embodiments, cancer cells may be treated by methods andcompositions of the embodiments. Cancer cells that may be treated withcell targeting constructs according to the embodiments include but arenot limited to cells from the bladder, blood, bone, bone marrow, brain,breast, colon, esophagus, gastrointestine, gum, head, kidney, liver,lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue,or uterus. In addition, the cancer may specifically be of the followinghistological type, though it is not limited to these: neoplasm,malignant; carcinoma; carcinoma, undifferentiated; giant and spindlecell carcinoma; small cell carcinoma; papillary carcinoma; squamous cellcarcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrixcarcinoma; transitional cell carcinoma; papillary transitional cellcarcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma;hepatocellular carcinoma; combined hepatocellular carcinoma andcholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma;adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposiscoli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolaradenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clearcell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma;papillary and follicular adenocarcinoma; nonencapsulating sclerosingcarcinoma; adrenal cortical carcinoma; endometroid carcinoma; skinappendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma;ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma;papillary cystadenocarcinoma; papillary serous cystadenocarcinoma;mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cellcarcinoma; infiltrating duct carcinoma; medullary carcinoma; lobularcarcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cellcarcinoma; adenosquamous carcinoma; adenocarcinoma w/squamousmetaplasia; thymoma, malignant; ovarian stromal tumor, malignant;thecoma, malignant; granulosa cell tumor, malignant; androblastoma,malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipidcell tumor, malignant; paraganglioma, malignant; extra-mammaryparaganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignantmelanoma; amelanotic melanoma; superficial spreading melanoma; maligmelanoma in giant pigmented nevus; epithelioid cell melanoma; bluenevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma,malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma;embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma;mixed tumor, malignant; mullerian mixed tumor; nephroblastoma;hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor,malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma,malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant;struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant;hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma;hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma;juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant;mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma;odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma,malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma;glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma;fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma;oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactoryneurogenic tumor; meningioma, malignant; neurofibrosarcoma;neurilemmoma, malignant; granular cell tumor, malignant; malignantlymphoma; hodgkin's disease; hodgkin's; paragranuloma; malignantlymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse;malignant lymphoma, follicular; mycosis fungoides; other specifiednon-hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mastcell sarcoma; immunoproliferative small intestinal disease; leukemia;lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcomacell leukemia; myeloid leukemia; basophilic leukemia; eosinophilicleukemia; monocytic leukemia; mast cell leukemia; megakaryoblasticleukemia; myeloid sarcoma; and hairy cell leukemia.

In preferred embodiments systemic formulations of the cell targetingconstructs are contemplated. Systemic formulations include thosedesigned for administration by injection, e.g. subcutaneous,intravenous, intramuscular, intrathecal or intraperitoneal injection, aswell as those designed for transdermal, transmucosal, inhalation, oralor pulmonary administration. In the most preferred embodiments celltargeted serine protease is delivered by direct intravenous orintratumoral injection.

For injection, the proteins of the embodiments may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hanks' solution, Ringer's solution, or physiological saline buffer.The solution may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Alternatively, the proteins may be in powder form for constitution witha suitable vehicle, e.g., sterile pyrogen-free water, before use.

A. Effective Dosages

The cell targeted serine protease of the embodiments will generally beused in an amount effective to achieve the intended purpose. For use totreat or prevent a disease condition, the molecules of the embodiments,or pharmaceutical compositions thereof, are administered or applied in atherapeutically effective amount. A therapeutically effective amount isan amount effective to ameliorate or prevent the symptoms, or prolongthe survival of, the patient being treated. Determination of atherapeutically effective amount is well within the capabilities ofthose skilled in the art, especially in light of the detailed disclosureprovided herein.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays. For example, a dose can beformulated in animal models to achieve a circulating concentration rangethat includes the IC₅₀ as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g., animalmodels, using techniques that are well known in the art. One havingordinary skill in the art could readily optimize administration tohumans based on animal data.

Dosage amount and interval may be adjusted individually to provideplasma levels of the molecules which are sufficient to maintaintherapeutic effect. Usual patient dosages for administration byinjection range from about 0.1 to 5 mg/kg/day, preferably from about 0.5to 1 mg/kg/day. Therapeutically effective serum levels may be achievedby administering multiple doses each day.

In cases of local administration or selective uptake, the effectivelocal concentration of the proteins may not be related to plasmaconcentration. One having skill in the art will be able to optimizetherapeutically effective local dosages without undue experimentation.

The amount of molecules administered will, of course, be dependent onthe subject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgment of theprescribing physician.

The therapy may be repeated intermittently while symptoms detectable oreven when they are not detectable. The therapy may be provided alone orin combination with other drugs. In the case of autoimmune disorders,the drugs that may be used in combination with serine proteaseconstructs of the embodiments include, but are not limited to, steroidand non-steroid anti-inflammatory agents.

B. Toxicity

Preferably, a therapeutically effective dose of the cell targeted GrBdescribed herein will provide therapeutic benefit without causingsubstantial toxicity.

Toxicity of the molecules described herein can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., by determining the LD₅₀ (the dose lethal to 50% of the population)or the LD₁₀₀ (the dose lethal to 100% of the population). The dose ratiobetween toxic and therapeutic effect is the therapeutic index. Proteinswhich exhibit high therapeutic indices are preferred. The data obtainedfrom these cell culture assays and animal studies can be used informulating a dosage range that is not toxic for use in human. Thedosage of the proteins described herein lies preferably within a rangeof circulating concentrations that include the effective dose withlittle or no toxicity. The dosage may vary within this range dependingupon the dosage form employed and the route of administration utilized.The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition. (See,e.g., Fingl et al., 1975).

C. Pharmaceutical Preparations

Pharmaceutical compositions of the present embodiments comprise aneffective amount of one or more chimeric polypeptides or chimericpolypeptides and at least one additional agent dissolved or dispersed ina pharmaceutically acceptable carrier. The phrases “pharmaceutical orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce an adverse, allergic or other untowardreaction when administered to an animal, such as, for example, a human,as appropriate. The preparation of an pharmaceutical composition thatcontains at least one chimeric polypeptide or additional activeingredient will be known to those of skill in the art in light of thepresent disclosure, as exemplified by Remington's PharmaceuticalSciences, 18th Ed. Mack Printing Company, 1990, incorporated herein byreference. Moreover, for animal (e.g., human) administration, it will beunderstood that preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the therapeutic orpharmaceutical compositions is contemplated.

The cell targeted serine protease may comprise different types ofcarriers depending on whether it is to be administered in solid, liquidor aerosol form, and whether it need to be sterile for such routes ofadministration as injection. The present therapies of the embodimentscan be administered intravenously, intradermally, intraarterially,intraperitoneally, intralesionally, intracranially, intraarticularly,intraprostaticaly, intrapleurally, intratracheally, intranasally,intravitreally, intravaginally, intrarectally, topically,intratumorally, intramuscularly, intraperitoneally, subcutaneously,subconjunctival, intravesicularlly, mucosally, intrapericardially,intraumbilically, intraocularally, orally, topically, locally,inhalation (e.g., aerosol inhalation), injection, infusion, continuousinfusion, localized perfusion bathing target cells directly, via acatheter, via a lavage, in cremes, in lipid compositions (e.g.,liposomes), or by other method or any combination of the forgoing aswould be known to one of ordinary skill in the art (see, for example,Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference).

The actual dosage amount of a composition of the present embodimentsadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. The practitioner responsible for administration will, inany event, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. In other non-limitingexamples, a dose may also comprise from about 5 mg/kg/body weight toabout 100 mg/kg/body weight, about 5 microgram/kg/body weight to about500 milligram/kg/body weight, etc., can be administered, based on thenumbers described above.

In any case, the composition may comprise various antioxidants to retardoxidation of one or more component. Additionally, the prevention of theaction of microorganisms can be brought about by preservatives such asvarious antibacterial and antifungal agents, including but not limitedto parabens (e.g., methylparabens, propylparabens), chlorobutanol,phenol, sorbic acid, thimerosal or combinations thereof.

In embodiments where compositions are provided in a liquid form, acarrier can be a solvent or dispersion medium comprising but not limitedto, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquidpolyethylene glycol, etc.), lipids (e.g., triglycerides, vegetable oils,liposomes) and combinations thereof. The proper fluidity can bemaintained, for example, by the use of a coating, such as lecithin; bythe maintenance of the required particle size by dispersion in carrierssuch as, for example liquid polyol or lipids; by the use of surfactantssuch as, for example hydroxypropylcellulose; or combinations thereofsuch methods. In many cases, it will be preferable to include isotonicagents, such as, for example, sugars, sodium chloride or combinationsthereof.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and/or the otheringredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, the preferredmethods of preparation are vacuum-drying or freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessary andthe liquid diluent first rendered isotonic prior to injection withsufficient saline or glucose. The preparation of highly concentratedcompositions for direct injection is also contemplated, where the use ofDMSO as solvent is envisioned to result in extremely rapid penetration,delivering high concentrations of the active agents to a small area.

The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectablecomposition can be brought about by the use in the compositions ofagents delaying absorption, such as, for example, aluminum monostearate,gelatin or combinations thereof.

VI. Combination Therapies

In order to increase the effectiveness of a nucleic acid, polypeptide ornanoparticle complex of the present embodiments, it may be desirable tocombine these compositions with other agents effective in the treatmentof the disease of interest.

As a non-limiting example, the treatment of cancer may be implementedwith a cell-targeted serine protease therapeutic of the presentembodiments along with other anti-cancer agents. An “anti-cancer” agentis capable of negatively affecting cancer in a subject, for example, bykilling cancer cells, inducing apoptosis in cancer cells, reducing thegrowth rate of cancer cells, reducing the incidence or number ofmetastases, reducing tumor size, inhibiting tumor growth, reducing theblood supply to a tumor or cancer cells, promoting an immune responseagainst cancer cells or a tumor, preventing or inhibiting theprogression of cancer, or increasing the lifespan of a subject withcancer. More generally, these other compositions would be provided in acombined amount effective to kill or inhibit proliferation of the cell.This process may involve contacting the cells with the anti-cancerpeptide or nanoparticle complex and the agent(s) or multiple factor(s)at the same time. This may be achieved by contacting the cell with asingle composition or pharmacological formulation that includes bothagents, or by contacting the cell with two distinct compositions orformulations, at the same time, wherein one composition includes theanti-cancer peptide or nanoparticle complex and the other includes thesecond agent(s). In particular embodiments, an anti-cancer peptide canbe one agent, and an anti-cancer nanoparticle complex can be the otheragent.

Treatment with the anti-cancer peptide or nanoparticle-complex mayprecede or follow the other agent treatment by intervals ranging fromminutes to weeks. In embodiments where the other agent and theanti-cancer peptide or nanoparticle complex are applied separately tothe cell, one would generally ensure that a significant period of timedid not expire between the time of each delivery, such that the agentand the anti-cancer peptide or nanoparticle complex would still be ableto exert an advantageously combined effect on the cell. In suchinstances, it is contemplated that one may contact the cell with bothmodalities within about 12-24 hours of each other and, more preferably,within about 6-12 hours of each other. In some situations, it may bedesirable to extend the time period for treatment significantly whereseveral days (e.g., 2, 3, 4, 5, 6 or 7 days) to several weeks (e.g., 1,2, 3, 4, 5, 6, 7 or 8 weeks) lapse between the respectiveadministrations.

Various combinations may be employed, where the serine protease-basedtherapy is “A” and the secondary agent, such as radiotherapy,chemotherapy or anti-inflammatory agent, is “B”:

-   -   A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B    -   B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A    -   B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

In certain embodiments, administration of the GRB therapy of the presentembodiments to a patient will follow general protocols for theadministration of chemotherapeutics, taking into account the toxicity,if any, of the vector. It is expected that the treatment cycles would berepeated as necessary. It also is contemplated that various standardtherapies, as well as surgical intervention, may be applied incombination with the described hyperproliferative cell therapy.

A. Chemotherapy

Cancer therapies also include a variety of combination therapies. Insome aspects a serine protease therapeutic of the embodiments isadministered (or formulated) in conjunction with a chemotherapeuticagent. For example, in some aspects the chemotherapeutic agent is aprotein kinase inhibitor such as a EGFR, VEGFR, AKT, Erb1, Erb2, ErbB,Syk, Bcr-Abl, JAK, Src, GSK-3, PI3K, Ras, Raf, MAPK, MAPKK, mTOR, c-Kit,eph receptor or BRAF inhibitors. Nonlimiting examples of protein kinaseinhibitors include Afatinib, Axitinib, Bevacizumab, Bosutinib,Cetuximab, Crizotinib, Dasatinib, Erlotinib, Fostamatinib, Gefitinib,Imatinib, Lapatinib, Lenvatinib, Mubritinib, Nilotinib, Panitumumab,Pazopanib, Pegaptanib, Ranibizumab, Ruxolitinib, Saracatinib, Sorafenib,Sunitinib, Trastuzumab, Vandetanib, AP23451, Vemurafenib, MK-2206,GSK690693, A-443654, VQD-002, Miltefosine, Perifosine, CAL101, PX-866,LY294002, rapamycin, temsirolimus, everolimus, ridaforolimus, Alvocidib,Genistein, Selumetinib, AZD-6244, Vatalanib, P1446A-05, AG-024322,ZD1839, P276-00, GW572016 or a mixture thereof.

Yet further combination chemotherapies include, for example, alkylatingagents such as thiotepa and cyclosphosphamide; alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammalI and calicheamicinomegaI1; dynemicin, including dynemicin A; bisphosphonates, such asclodronate; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antiobiotic chromophores, aclacinomysins,actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin,carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin(including morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues such as denopterin,pteropterin, trimetrexate; purine analogs such as fludarabine,6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such asancitabine, azacitidine, 6-azauridine, carmofur, cytarabine,dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens suchas calusterone, dromostanolone propionate, epitiostanol, mepitiostane,testolactone; anti-adrenals such as mitotane, trilostane; folic acidreplenisher such as frolinic acid; aceglatone; aldophosphamideglycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil;bisantrene; edatraxate; defofamine; demecolcine; diaziquone;elformithine; elliptinium acetate; an epothilone; etoglucid; galliumnitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such asmaytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharidecomplex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid;triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especiallyT-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, e.g.,paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine;platinum coordination complexes such as cisplatin, oxaliplatin andcarboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;mitoxantrone; vincristine; vinorelbine; novantrone; teniposide;edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan(e.g., CPT-11); topoisomerase inhibitor RFS 2000;difluorometlhylornithine (DMFO); retinoids such as retinoic acid;capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien,navelbine, farnesyl-protein tansferase inhibitors, transplatinum, andpharmaceutically acceptable salts, acids or derivatives of any of theabove. In certain embodiments, the compositions provided herein may beused in combination with gefitinib. In other embodiments, the presentembodiments may be practiced in combination with Gleevac (e.g., fromabout 400 to about 800 mg/day of Gleevac may be administered to apatient). In certain embodiments, one or more chemotherapeutic may beused in combination with the compositions provided herein.

B. Radiotherapy

Radiotherapy has been used extensively in treatments and includes whatare commonly known as γ-rays, X-rays, and/or the directed delivery ofradioisotopes to tumor cells. Other forms radiotherapy are alsocontemplated such as microwaves and UV-irradiation. It is most likelythat all of these factors effect a broad range of damage on DNA, on theprecursors of DNA, on the replication and repair of DNA, and on theassembly and maintenance of chromosomes. Dosage ranges for X-rays rangefrom daily doses of 50 to 200 roentgens for prolonged periods of time (3to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges forradioisotopes vary widely, and depend on the half-life of the isotope,the strength and type of radiation emitted, and the uptake by theneoplastic cells.

The terms “contacted” and “exposed,” when applied to a cell, are usedherein to describe the process by which a therapeutic composition and achemotherapeutic or radiotherapeutic agent are delivered to a targetcell or are placed in direct juxtaposition with the target cell. Toachieve cell killing or stasis, both agents are delivered to a cell in acombined amount effective to kill the cell or prevent it from dividing.

C. Immunotherapy

Immunotherapeutics, generally, rely on the use of immune effector cellsand molecules to target and destroy cancer cells. The immune effectormay be, for example, an antibody specific for some marker on the surfaceof a tumor cell. The antibody alone may serve as an effector of therapyor it may recruit other cells to actually affect cell killing. Theantibody also may be conjugated to a drug or toxin (chemotherapeutic,radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) andserve merely as a targeting agent. Alternatively, the effector may be alymphocyte carrying a surface molecule that interacts, either directlyor indirectly, with a tumor cell target. Various effector cells includecytotoxic T cells and NK cells.

Immunotherapy, thus, could be used as part of a combined therapy, inconjunction with a serine protease therapy of the present embodiments.The general approach for combined therapy is discussed below. Generally,the tumor cell must bear some marker that is amenable to targeting,i.e., is not present on the majority of other cells. Many tumor markersexist and any of these may be suitable for targeting in the context ofthe present embodiments. Common tumor markers include carcinoembryonicantigen, prostate specific antigen, urinary tumor associated antigen,fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl LewisAntigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb Band p155.

D. Gene Therapy

In yet another embodiment, the secondary treatment is a gene therapy inwhich a therapeutic polynucleotide is administered before, after, or atthe same time as the therapeutic composition. Viral vectors for theexpression of a gene product are well known in the art, and include sucheukaryotic expression systems as adenoviruses, adeno-associated viruses,retroviruses, herpesviruses, lentiviruses, poxviruses including vacciniaviruses, and papiloma viruses, including SV40. Alternatively, theadministration of expression constructs can be accomplished with lipidbased vectors such as liposomes or DOTAP:cholesterol vesicles. All ofthese method are well known in the art (see, e.g. Sambrook et al., 1989;Ausubel et al., 1998; Ausubel, 1996).

Delivery of a vector encoding one of the following gene products willhave a combined anti-hyperproliferative effect on target tissues. Avariety of proteins are encompassed within the present embodiments andare well known in the art.

E. Surgery

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative andpalliative surgery. Curative surgery is a cancer treatment that may beused in conjunction with other therapies, such as the treatmentsprovided herein, chemotherapy, radiotherapy, hormonal therapy, genetherapy, immunotherapy and/or alternative therapies.

Curative surgery includes resection in which all or part of canceroustissue is physically removed, excised, and/or destroyed. Tumor resectionrefers to physical removal of at least part of a tumor. In addition totumor resection, treatment by surgery includes laser surgery,cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs'surgery). It is further contemplated that the present embodiments may beused in conjunction with removal of superficial cancers, precancers, orincidental amounts of normal tissue.

Upon excision of part of all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

VII. Examples

The following examples are included to demonstrate preferred embodimentsof the embodiments. It should be appreciated by those of skill in theart that the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the embodiments, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1—Testing of GrB-VEGF Fusion Constructs

Four different fusion constructs were generated using wild-type (native)human GrB (WT), a mutant with the potential self-cleavage domain deleted(A), a mutant with one glycosylation domain mutated (N1) and a versioncombining the two mutations (A, N1). The constructs were generated byPCR, the mutations were confirmed by DNA sequencing and the proteinswere transfected into mammalian expression cells. The proteins wereexpressed and purified. In vitro assays with the expressed polypeptidesshow similar levels for enzymatic activity (FIG. 3). The fusion proteinswere then used to treat transfected endothelial cells expressing theVEGFR-2 receptor (PAE/VEGFR-2) or a control (PAE/VEGFR-1) cell line.Results of the studies are shown in Table 1. Values shown are the IC₅₀values in nM.

TABLE 1 Cytotoxic effects of GrB/VEGF₁₂₁ fusion construct variants ontransfected endothelial cells. Target cell WT A N1 A, N1 PAE/ 15.3 12.211.6 19.6 VEGFR-2 (+) PAE/ >1000 >1000 >1000 >1000 VEGFR-1 (−) valuesindicate IC₅₀ nM.

These studies showed that the above modifications had no effect onoverall expression/yield of Granzyme B fusion proteins. The enzymaticand cytotoxic activity of the Granzyme B C210A mutation was similar tonative Granzyme B. Thus, this mutant is a better candidate for GranzymeB chemical conjugation studies than native Granzyme B.

Example 2—Investigation of Linker Effects on GrB Activity

GrB-ZME(VL-VH) fusion proteins were constructed as indicated above usingdifferent linkers between the GrB and ZME sequences. The construct SLused the G₄S linker; LL was four repeats of the HL (EAAAK) linker and Xwas a G₄S+218 linker. All linkers showed specific cytotoxic effectsagainst target cells. The construct containing the shortest flexiblelinker demonstrated the best cytotoxicity (lowest IC₅₀) against targetcells as shown in Table 2. Thus, the studies indicate that short linkersmay produce more effective therapeutics.

TABLE 2 Cytotoxic effects of GrB/ZME fusion constructs relative totarget cells. AAB-527 and A375-M were specifically targeted by ZME,whereas SKOV3 was a non-specific control. Target cell SL LL X GrB aloneAAB-527 544 817 1148 >2000 A375-M 722 1124 2228 >2000 SKOV3 795 16672363 >2000 values indicate IC₅₀ (nM)

Example 3—Effect of GrB Glycosylation on Targeting Construct Activity

Two glycosylation sites were identified within the GrB molecule (d1 andd2) and modified the GrB/scFvMEL fusion construct as detailed above andas shown in FIG. 4 (d1 indicates N51S; d2 indicates N84A). Eachglycosylation site was modified and then a molecule containing bothmodifications was generated. The individual modifications had littleeffect on in vitro GrB enzymatic activity (see, e.g., FIG. 4). However,as shown in Table 3, removal of each of the glycosylation sitesgenerated a molecule with a lower IC₅₀ than the originalwild-type-containing GrB. There was little impact on the non-specificcell line (SKOV3).

TABLE 3 Cytotoxic effects of GrB/ZME fusion constructs relative totarget cells. SL is WT GrB, SL-1 includes the d1 mutation; SL-2 includesthe d2 mutation; and SL-3 is d1 and d2. AAB-527 and A375-M werespecifically targeted by ZME, whereas SKOV3 was a non-specific control.Target cell SL SL-1 SL-2 SL-3 AAB-527 544 259 291 * A375-M 722 216 438 *SKOV3 795 869 801 * * values not determined due to low yield. valuesindicate IC₅₀ (nM)

Example 4—Effect of GrB Glycosylation and T1 Translocation Domain onTargeting Construct Activity

Three expression constructs were developed and tested. (LL) encodedGrB-HL-HL-HL-HL-ZME(VL-VH), plasmid designation pSECTag-GrB-HL4-ZME; (E)encoded GrB-HL-HL-HL-HL-T1-ZME(VL-VH), plasmid designationpSECTag-GrB-HL4-T1-ZME; and (J) (LL) encoded GrB (d1, A,N)-HL-HL-HL-HL-ZME(VL-VH), plasmid designation pSECTag-GrB-HL4-ZME. Theconstructs were expressed and tested. Results are shown in Table 4 anddemonstrate that incorporation of the T1 domain increased the specificcytotoxicity of the construct against target cells but had no impact onnon-specific toxicity. Incorporation of d1 modified GrB into theconstruct further increased the specific cytotoxicity of the constructwith no impact on non-specific cytotoxicity. This result wasparticularly evident in the case of the constructs including the GrB(d1, A, N) polypeptide.

TABLE 4 Cytotoxic effects of various GrB/ZME fusion constructs with andwithout the T1 translocation domain. Target cell LL E J AAB-527 817 401227 A375-M 1124 700 265 SKOV3 1667 1765 1603 values indicate IC₅₀ (nM).

Example 5—Effect of Linker Designs, Translocation Domains and/orEndosomal Cleavable Peptides on Targeting Construct Activity

Further GrB expression constructs were developed and tested. (LL)encoded GrB-HL-HL-HL-HL-ZME(VL-VH), plasmid designationpSECTag-GrB-HL4-ZME; (E) encoded GrB-HL-HL-HL-HL-T1-ZME(VL-VH), plasmiddesignation pSECTag-GrB-HL4-T1-ZME; (M1) encodedGrB-HL-HL-HL-HL-T1-Fur-ZME(VL-VH), plasmid designationpSECTag-GrB-HL4-T1-Fur-ZME; (X) encoded GrB-G₄S/218-ZME(VL-VH), plasmiddesignation pSECTag-GrB-G218-ZME; (W) encodedGrB-G₄S/218-INF7-GSGSG-ZME(VL-VH), plasmid designationpSECTag-GrB-G218-INF7-ZME and (WF) encodedGrB-G₄S/218-INF7-Fur-GSGSG-ZME(VL-VH), plasmid designationpSECTag-GrB-G218-INF7-Fur-ZME.

These constructs were expressed and tested. Results shown in Table 5demonstrate that addition of the Furin-cleavable site to the “E”molecule (M1) increased the sensitivity of both specific andnon-specific cell lines. Incorporation of the INF7 peptide to improvetranslocation across the membrane greatly increased the sensitivity oftarget cells (constructs W and WF).

TABLE 5 Cytotoxic effects of various GrB/ZME fusion constructs withvarious linkers, translocation domains and cleavage sites. Target cellLL E M1 X W WF AAB-527 817 401 253 1148 13 119 A375-M 1124 700 448 2228134 279 SKOV3 1667 1765 936 2363 102 663 values indicate IC₅₀ (nM).

Example 6—Effect of C-Terminal Translocation Domains on TargetingConstruct Activity

Further GrB expression constructs were developed and tested. (LL)encoded GrB-HL-HL-HL-HL-ZME(VL-VH), plasmid designationpSECTag-GrB-HL4-ZME; (F) encoded GrB-HL-HL-HL-HL-ZME(VL-VH)-penetratin,plasmid designation pSECTag-GrB-HL4-ZME-Penetratin; and (T) encodedGrB-G₄S/218-ZME(VL-VH)-218-26, plasmid designationpSECTag-GrB-G218-ZME-26.

These constructs were expressed and tested. Results shown in Table 6demonstrate that incorporation of Penetratin had no impact on thebiological activity of the fusion construct. Incorporation of the “26”molecule increased the toxicity to target and non-target cells alike.

TABLE 6 Cytotoxic effects of various GrB/ZME fusion constructs withvarious C-terminal translocation domains. Target cell LL F T AAB-527 817849 78 A375-M 1124 1159 24 SKOV3 1667 1366 90 values indicate IC₅₀ (nM).

Example 7—Effect of Different Membrane Translocation Peptides in theSame Relative Position on Targeting Construct Activity

Further GrB expression constructs were developed and tested. (U) encodedGrB-G₄S/218-26-GSGSG-ZME(VL-VH), plasmid designationpSECTag-GrB-G218-26-ZME; (Y) encoded GrB-G₄S/218-T1-GSGSG-ZME(VL-VH),plasmid designation pSECTag-GrB-G218-T1-ZME; (YY) encodedGrB-G₄S/218-T2-GSGSG-ZME(VL-VH), plasmid designationpSECTag-GrB-G218-T2-ZME; and (W) encodedGrB-G₄S/218-INF7-GSGSG-ZME(VL-VH), plasmid designationpSECTag-GrB-G218-INF7-ZME.

These constructs were expressed and tested. Results shown in Table 7demonstrate that constructs containing “26,” T1, and T2 (U, Y and YY)were less toxic to target cells than to the non-specific cell line. Onlyconstruct W containing the INF7 membrane translocation peptide showedclear improvement in specificity.

TABLE 7 Cytotoxic effects of various GrB/ZME fusion constructs withvarious translocation domains. Target cell U Y YY W AAB-527 131486 >2000 13 A375-M 29 364 240 134 SKOV3 64 364 363 102 values indicateIC₅₀ (nM).

Example 8—Specificity of Targeting Construct with and without MembraneTranslocation Peptides

Further GrB expression constructs were developed and tested. (GrB)encoded GrB, plasmid designation pSECTag-GrB; (GrB-26) encodedGrB-G₄S/218-26, plasmid designation pSECTag-GrB-G218-26; (GrB-4D5)encoded GrB-G₄S/218-4D5(V1-VH), plasmid designation pSECTag-GrB-4D5; and(GrB-4D5-26) encoded GrB-G₄S/218-4D5(VL-VH)-218-26, plasmid designationpSECTag-GrB-4D5-26.

These constructs were expressed and tested. Results shown in Table 8demonstrate that the GrB/4D5 construct was not active on HER2 expressingtarget cells. Incorporation of the “26” translocation peptide restoredsensitivity to HER2 positive cells but did not increase the cytotoxicityto HER2 negative cells.

TABLE 8 Cytotoxic effects of various GrB/4D5 fusion constructs with andwithout a translocation domain. Her2 Exp. Cell line GrB-4D5-26 GrB-4D5GrB-26 GrB High BT-474-M1 33 >200 >200 >1000 High BT-474-27 >200 >200 >1000 M1(HR) High Calu-3 10 96 >200 >1000 High NCI-N8787 >200 >200 >1000 High MDA-MB- 25 >200 >200 >1000 453 HighSKBR3 >200 >200 >200 >1000 High SKOV3 >200 >200 >200 >1000 noneMe-180 >200 >200 >200 >1000 values indicate IC₅₀ (nM).

Example 9—Effect of an Endosomal Cleavage Peptide (ECP) on TargetingConstruct Activity

Further GrB expression constructs were developed and tested. (XF)encoded GrB-G₄S/218-Fur-GSGSG-ZME(VL-VH), plasmid designationpSECTag-GrB-G218-ZME; (UF) encoded GrB-G₄S/218-26-Fur-GSGSG-ZME(VL-VH),plasmid designation pSECTag-GrB-G218-26-Fur-ZME; (YF) encodedGrB-G₄S/218-T1-Fur-GSGSG-ZME(VL-VH), plasmid designationpSECTag-GrB-G218-T1-Fur-ZME; (WF) encodedGrB-G₄S/218-INF7-Fur-GSGSG-ZME(VL-VH), plasmid designationpSECTag-GrB-G218-INF7-ZME; and (ZF) encodedGrB-SSG-CCP-MTP-Fur-GSGSG-ZME(VL-VH), plasmid designationpSECTag-GrB-Ad2-ZME.

These constructs were expressed and tested. Results shown in Table 9.

TABLE 9 Cytotoxic effects of various GrB fusion constructs with furincleavage sites. Target cell XF YF WF ZF UF AAB-527 166 95 119 88 *A375-M 657 1057 279 164 * SKOV3 1547 1429 663 328 * values indicate IC₅₀nM. * values not determined due to low yield

Example 10—Assessment of Cytotoxic Activity of Further GrB FusionConstructs

Further GrB expression constructs were developed and tested. (GrB)encoded GrB, plasmid designation pSECTag-GrB. (GrB-TWEAK) encodedGrB-G₄S-TNF-like weak inducer of apoptosis (TWEAK). These constructswere expressed and tested. Results of cytotoxicity studies are shown inTable 10.

TABLE 10 Cytotoxic effects of GrB versus GrB-TWEAK in various celllines. Cell line GrB-TWEAK GrB MDA-MB435/MDR1 0.4 447 MDA-MB435 11 445AAB-527 4 1044 SK-Mel-5 33 3014 WM35 77 >1923 SB2 219 >1923 A375-M226 >1923 SK-Mel-1 330 >1923 SK-Mel-28 1720 >1923 MDA-MB231 15 >1923SKBR3 64 660 MCF-7 307 >1923 ES-2 67 1435 OC-316 89 >1923 HeyA8 108 841HeyA8-MDR 100 >700 A2780 263 >1297 HEY 271 1015 T-24 29 2631 HT-29 23800 A172 55 1911 HT-1080 72 1297 BxPC-3 239 >1923 U87MG 144 825Jurkat >700 >700 values indicate IC₅₀ nM.

Example 11—Construction and Characterization of GrB Fusions TargetingHer2

Cell Lines and Cultures.

The cell lines BT474 M1, NCI-N87, Calu3, MDA MB435, and Me180 were allobtained from American Type Culture Collection (Manassas, Va.). Thehuman breast cancer cell line MDA MB453 was generously supplied by Dr.Zhen Fan (The University of Texas MD Anderson Cancer Center, Houston,Tex.). The human breast cancer cell line eB-1 was kindly provided by Dr.Dihua Yu (The University of Texas MD Anderson Cancer Center, Houston,Tex.). BT474 M1 HR and LR cells were derived from BT474 M1 cells after a12-month selection in the continuous presence of 1 μM Herceptin or 1.5μM Lapatinib. BT474 M1 MDR-1 cells were generated by the transfection ofplasmid pHaMDR1 to parental BT474 M1 cells. The HEK 293T cell line wassupplied by Dr. Bryant G. Darnay (MD Anderson Cancer Center). All celllines were maintained in Dulbecco's Modified Eagle Medium or RPMI 1640medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mML-glutamine, and 1 mM antibiotics.

Construction, Expression, and Purification of GrB-Based Fusions.

The sequence of the humanized anti-Her2/neu scFv 4D5 was derived fromthe published Herceptin light- and heavy-chain variable domain sequences(Carter et al., 1992). Previous observations suggested that use offusogenic peptides facilitates endosomal escape and delivery of largemolecules into the cytosol (Plank et al., 1994; Bongartz et al., 1994).Therefore, the inventors incorporated the fusogenic peptide 26.

The GrB/4D5/26, GrB/4D5, GrB/26, and GrB DNA constructs were generatedby an overlapping polymerase chain reaction method. Illustrations of theconstructs are shown in FIG. 5A. The inventors designed a universal 218linker (GSTSGSGKPGSGEGSTKG; SEQ ID NO: 13) incorporated between theindividual components of GrB, 4D5 (SEQ ID NO: 23), or peptide 26.Peptide 26 (AALEALAEALEALAEALEALAEAAAA; SEQ ID NO: 22) was generatedfrom the 29-residue amphipathic peptide without the three C-terminalamino acids, which was responsible for dimerization (Turk et al., 2002).All construct genes were cloned into the mammalian cell expressionvector pSecTag (Life Technologies, Carlsbad, Calif.).

A total of 3×10⁷ HEK 293T cells were transfected using 50 μg of plasmidDNA and 150 μL (1 mg/mL) of polyethylenimine reagent, which were addedto OPTI-MEM medium (Life Technologies) and incubated for 20 min at roomtemperature before the transfection mixture was added to the cells.After overnight incubation at 37° C., 100% humidity, and 5% CO₂, DMEMserum-free medium was added and the cells were incubated for a further 3days. GrB-based protein samples were purified from cell culturesupernatants by immobilized metal affinity chromatography, as previouslyreported (Cao et al., 2009). Activation of the protein was achieved byovernight incubation with recombinant enterokinase (Merck, WhitehouseStation, N.J.) according to the manufacturer's instructions. Afterdialysis against phosphate-buffered saline, the proteins were filtersterilized and stored at −80° C.

GrB-based fusions were generated by fusing GrB to 4D5 with (designatedGrB/4D5/26; SEQ ID NO: 24) or without (designated GrB/4D5) the additionof pH-sensitive fusogenic peptide 26 to the C-terminal of the construct.Furthermore, GrB and GrB/26 were used as controls. All fusion proteinswere expressed in human embryonic kidney cells (HEK 293T). Followingpurification, the final products migrated at the expected molecularweights, with a purity of >95% (FIG. 5B).

Analysis of Binding Affinity.

The K_(d) value and specificity of GrB-based protein samples wereevaluated by ELISA. Rabbit anti-c-myc antibody and horseradishperoxidase-conjugated goat anti-rabbit immunoglobulin G were used astracers in this assay, as described previously (Cao et al., 2012).

The binding affinities (K_(d) values) of GrB/4D5/26 and GrB/4D5 wereassessed by ELISA using purified Her2/neu extracellular domain (ECD),Her2/neu-positive BT474 M1 human breast cancer cells, andHer2/neu-negative Me180 human cervical cancer cells. Both fusionsspecifically bound to Her2/neu ECD and BT474 M1 cells but not to Me180cells (FIG. 6A). The apparent K_(d) values were determined bycalculating the concentration of fusion constructs that producedhalf-maximal specific binding. GrB/4D5 and GrB/4D5/26 demonstratedapparent K_(d) values of 0.329 nM and 0.469 nM, respectively, toHer2/neu ECD and 0.383 nM and 0.655 nM, respectively, to BT474 M1 cells.These results are in general agreement with the published K_(d) valuefor native Herceptin to the Her2/neu receptor (0.15 nM) (Carter et al.,1992).

Enzymatic Assay of GrB-Based Fusions.

The enzymatic activity of the GrB component was determined in acontinuous colorimetric assay usingN-α-t-butoxycarbonyl-L-alanyl-L-alanyl-L-aspartyl-thiobenzylester(BAADT) as a specific substrate (Liu et al., 2003). Assays consisted ofcommercial human GrB (Enzyme Systems Products, Livermore, Calif.) orGrB-based fusion proteins in BAADT at 25° C. The change in absorbance at405 nm was measured on a Thermomax plate reader (Columbia, Md.).Increases in sample absorbance were converted to enzymatic rates byusing an extinction coefficient of 13,100 cm⁻¹ M⁻¹ at 405 nm. Thespecific activity of GrB-based fusion proteins was calculated usingnative GrB as the standard.

To assess the biological activity of the GrB component of the fusions,the inventors compared the ability of the constructs to cleave thesubstrate BAADT with that of native, authentic GrB (FIG. 6B). GrB/4D5and GrB/4D5/26 had intact GrB enzymatic activity (1.54×10⁵ U/μmoL and1.57×10⁵ U/μmoL, respectively). These activities were comparable to thatof the native GrB standard (1.19×10⁵ U/μmoL). Because the pro-GrB fusionconstructs contain purification tags on the N-terminal end of GrB andrender the molecule enzymatically inactive, these proteins were unableto cause hydrolysis of BAADT.

Cellular Uptake and GrB Delivery of Fusion Constructs.

Immunofluorescence-based internalization studies were performed usingBT474 M1 and Me180 cells. Cells were treated with 25 nM GrB/4D5/26 for 4h and subjected to immunofluorescent staining with anti-GrB antibody(fluorescein isothiocyanate [FITC]-conjugated secondary antibody).Nuclei were counterstained with PI. Visualization of immunofluorescencewas performed with a Zeiss LSM510 confocal laser scanning microscopeZeiss LSM510 (Carl Zeiss, Thornwood, N.Y.).

The GrB moiety of both fusions was observed primarily in the cytosolafter treatment with a fusion protein in BT474 M1 cells but not in Me180cells (FIG. 6C), demonstrating that both constructs were efficient incell binding and internalization after exposure to Her2/neu-positivecells. The internalization efficiency of the fusions was furtherexamined by time-dependent western blot analysis of the GrB signal(full-length GrB fusion+free GrB) (FIG. 6D). Both constructsinternalized rapidly into BT474 M1 cells within 30 min. Compared withGrB/4D5, GrB/4D5/26 displayed enhanced and long-lasting cellinternalization. The intracellular delivery of GrB after endocytosis ofGrB/4D5 or GrB/4D5/26 also was assessed by time-dependent westernblotting (free GrB). The inventors observed no GrB delivery by GrB/4D5up to 48 h of treatment, whereas GrB delivery by GrB/4D5/26 was observedstarting at approximately 4 h of treatment and presented a tremendouslyhigh level of free GrB up to 48 h (FIG. 6D).

In Vitro Cytotoxic Effects of GrB-Based Fusions.

Log-phase cells were seeded (˜5×10³ cells per well) in 96-well platesand allowed to attach overnight. Cells were further incubated withvarious concentrations of GrB-based fusion proteins, GrB, or medium at37° C. for 72 h. Cell viability was determined using the crystal violetstaining method followed by solubilization of the dye in Sorenson'sbuffer as described previously (Cao et al., 2009).

GrB-based fusions were tested against a number of tumor cell lines.After 72 h exposure, GrB/4D5/26 demonstrated specific cytotoxicity toHer2/neu-positive cells, with IC₅₀ values of less than 100 nM (Table11), and GrB/4D5 demonstrated cytotoxic effects at somewhat higher doses(>200 nM). In addition, GrB/26 showed minimal cytotoxicity at doses >600nM, but no significant activity of GrB itself was observed at doses upto 1.5 μM. When Her2/neu-positive MDA MB453 cells were pretreated withHerceptin (5 μM) for 6 h and then treated with GrB/4D5/26 for 72 h, thecytotoxicity of GrB/4D5/26 was reduced (FIG. 11), thereby demonstratinga requirement for antigen binding of the GrB/4D5/26 construct.

The inventors further investigated the expression levels of theendogenous proteinase inhibitor 9 (PI-9) in different tumor cells (FIG.12, Table 11). These studies failed to find an association between theresponse of cells to the cytotoxicity of the GrB constructs and theendogenous expression of PI-9. This may suggest that factors other thanPI-9 may account for the observed differences in GrB/4D5/26 cytotoxicityto Her2/neu expressing target cells.

TABLE 11 Comparative IC₅₀ values of GrB-baed fusion constructs againstvarious types of tumor cell lines. Her2/neu IC₅₀ (nM) Cell line Typelevel PI-9 level GrB/4D5/26 GrB/4D5 GrB/26 GrB BT474 Breast **** * 29.3253.3 905.5 >1500.0 M1 Calu3 Breast **** ***** 40.5 242.4 863.0 >1500.0NCI- Gastric **** * 90.4 629.0 1106.0 >1500.0 N87 MDA Lung *** * 56.8436.0 694.2 >1500.0 MB453 eB-1 Breast ** — 93.1 551.3 1134.5 >1500.0 MDABreast * — >500.0 >750.0 1031 >1500.0 MB435 Me180Cervical * * >500.0 >750.0 >1500.0 >1500.0

Cytotoxic Effects of GrB/4D5/26 Against Cells Resistant to Herceptin orLapatinib.

Acquired resistance to Herceptin or Lapatinib can be mediated byconcomitant upregulation of Her2/neu downstream signaling pathways oractivation of signaling through the estrogen receptor (ER) pathway (Wanget al., 2011). In this study, the inventors developed a model ofHerceptin-resistant (HR) and Lapatinib-resistant (LR) variants of BT474M1 cells. Parental BT474 M1 cells were readily sensitive to bothHerceptin (IC₅₀: 52.5 nM) and Lapatinib (IC₅₀: 34.7 nM) (Table 12). HRcells demonstrated resistance to Herceptin (IC₅₀: 10.1 μM, F.R.: 192)but remained sensitive to Lapatinib (IC₅₀: 32.4 nM). LR cells showedresistance to high micromolar concentrations of both Herceptin (IC₅₀:74.1 μM, F.R.: 1411) and Lapatinib (IC₅₀: 8.2 μM, F.R.: 237). As shownin Table 12, cells resistant to Herceptin demonstrated equivalentsensitivity to the GrB/4D5/26 construct (IC₅₀˜30 nM for both HR andparental BT474 M1 cells). For LR cells, the IC₅₀ was marginallyincreased (2-fold) compared to parental cells (66.1 nM vs. 32.9 nM,respectively).

The inventors also demonstrated that addition of epidermal growth factor(EGF) or neuregulin-1 (NRG-1) growth factor, but not β-estradiol, toBT474 M1 parental cells can circumvent the cellular cytotoxic responsesto Herceptin and Lapatinib. Seventy-two hours of pretreatment of BT474M1 cells with 20 ng/mL EGF or 50 ng/mL NRG-1 resulted in a 400˜500 foldincrease in resistance to Herceptin and a 16-fold increase in resistanceto Lapatinib (Table 12). However, treatment of these resistant cellsresulted in no cross-resistance to GrB/4D5/26 fusions compared withparental BT474 M1 cells.

A significant observation was that incubation of cells with GrB/4D5/26in the presence of chloroquine did not improve cytotoxicity toward thesecells (FIG. 13). This finding demonstrated that the fusogenic peptide 26efficiently releases GrB fusion proteins from intracellular vesicles,thereby allowing access to cytosolic GrB substrates and induction ofapoptosis.

TABLE 12 Cytotoxic effects of Her2/neu-targeted therapeutic agents onIC₅₀ values in BT474 M1 cells and resistant variants. IC₅₀ (nM) with(Fold Resistance)* BT474 M1 + BT474 M1 + BT474 M1 BT474 M1 BT474 M1 +NRG- β- Agent BT474 M1 HR LR EGF** 1*** estradiol**** Herceptin 52.5 (1)10100.5 (192) 74100.0 (1411) 26305.0 (501) 23033.0 (439) 74.1 (1)Lapatinib 34.7 (1) 32.4 (1) 8225.0 (237) 543.0 (16) 547.1 (16) 33.9 (1)GrB/4D5/26 32.9 (1) 26.8 (1) 66.1 (2) 21.7 (1) 18.1 (1) 31.3 (1) *FoldResistance (F.R.) represents IC₅₀ of agent on BT474 M1 resistantvariants/that on BT474 M1 parental cells. Cells were pretreated with**20 ng/mL EGF, ***50 ng/mL NRG-1, or ****10 ng/mL beta-estradiol for 72h before drug treatment.

Mechanistic Studies of GrB/4D5/26 Cytotoxicity.

The inventors conducted a panel of experiments to assess the potentialof GrB-based fusions to initiate the proteolytic cascade culminating inapoptosis of BT474 M1 parental, HR, and LR cells.

Annexin V/Propidium Iodide (PI) Staining.

The Annexin V/PI staining assay was used to quantitatively determine thepercentage of cells undergoing apoptosis after exposure to GrB/4D5/26.Cells were seeded onto 6-well plates (5×10⁵ cells per well) andincubated with 100 nM GrB/4D5/26 at 37° C. for 24 or 48 h. Aliquots ofcells were washed with phosphate-buffered saline and then incubated withAnnexin V-FITC antibody. PI solution was added at the end of theincubation, and the cells were analyzed immediately by flow cytometry.

GrB/4D5/26 induced apoptosis in BT474 M1 parental, HR, and LR cells, asindicated by the reduced viable population combined with greaterpopulations of early apoptosis (FIG. 7A). No apoptosis was induced by100 nM GrB/4D5 in any of these cells (FIG. 14). Her2/neu-negative Me180cells were not affected by either construct.

Activation of Caspases.

Western blot analysis was used to identify activation of caspases-3, and-9 as well as PARP cleavage. Treatment of BT474 M1 cells with GrB/4D5/26resulted to the cleavage of caspase 3, caspase 9, and PARP in all cells,but no activation occurred when cells were treated with GrB/4D5 (FIG.7B). Compared with BT474 M1 parental and HR cells, the activations ofcaspase-9, caspase-3, and PARP were delayed in LR cells, which coincidedwith the observed decreased cytotoxic effects.

The inventors further assessed the kinetics of PARP cleavage induced byGrB/4D5/26 on BT474 M1 parental, HR, and LR cells, and found thatcleavage occurred after 2 h of drug exposure for parental and HR cellsbut at 24 h for LR cells (FIG. 7C). In addition, in the presence of thepan-caspase inhibitor zVAD-fmk, PARP cleavage of GrB/4D5/26 waspartially inhibited in all cells. This finding is in agreement with amechanism relying on GrB activity for caspase-3 cleavage followed byPARP cleavage.

Impact on Mitochondrial Pathways.

After treatment with GrB/4D5 or GrB/4D5/26, cells were collected andresuspended with 0.5 mL of 1× cytosol extraction buffer mix (BioVision,Milpitas, Calif.) and then homogenized in an ice-cold glass homogenizer.The homogenate was centrifuged, and the supernatant was collected andlabeled as the cytosolic fraction. The pellet was resuspended in 0.1 mLof mitochondrial extraction buffer and saved as the mitochondrialfraction. Aliquots of each cytosolic and mitochondrial fraction wereanalyzed by western blotting with antibodies recognizing cytochrome cand Bax (Santa Cruz Biotechnology, Santa Cruz, Calif.). In addition,apoptosis was analyzed by western blot analysis using antibodiesrecognizing Bcl-2 and BID (Santa Cruz Biotechnology).

The inventors detected cell death induced by GrB/4D5/26 via severalmitochondrial-related pathways. In BT474 M1 parental, HR, and LR cells,GrB/4D5/26 treatment activated BID and downregulated the anti-apoptoticBcl-2 protein (FIG. 8A), and it triggered the release of cytochrome cfrom the mitochondria into the cytosol (FIG. 8B). Bax was normallypresent in both the cytosol and mitochondria of untreated cells.However, when the cells were treated with GrB/4D5/26, Bax was decreasedin cytosol and increased in mitochondria (FIG. 8B). As previouslydescribed, treatment for 24 h with GrB/4D5/26 was shown to activate themitochondrial pathway in both BT474 M1 parental and HR cells, but thisactivation was delayed in LR cells.

Effects of GrB Fusions on her- and ER-Associated Signaling Pathways.

After treatment, cell lysates were analyzed by western blotting withantibodies recognizing Her2/neu and phosphorylated (p)-mTOR (S2448)(Cell Signaling Technology, Danvers, Mass.) as well as p-Her2/neu(Tyr877), p-Her2/neu (Tyr 1221/1222), EGF receptor, p-EGF receptor(Thr845), Her3, p-Her3 (Tyr1328), IGF1 receptor, p-IGF1 receptor (Tyr1165/1166), ER, PR, Akt, p-Akt, ERK, p-ERK (Thr 177/Thr 160), PTEN,PI-9, and β-actin (all from Santa Cruz Biotechonology). Immunoreactiveproteins were visualized by enhanced chemiluminescence.

The inventors examined the mechanistic effects of the constructs on Her-and ER-related signaling events in BT474 M1 parental cells and theresistant variants. As shown in FIG. 15, cells resistant to Herceptinhad enhanced Her family receptor activity but reduced levels ofprogesterone receptor (PR) and PI-9. In contrast, in LR cells there wastotal downregulation of Her family receptor activity but higher levelsof ER, PR, and PI-9.

Cells treated with GrB/4D5 or GrB/4D5/26 demonstrated the effects onthese signaling pathways, corresponding to the cytotoxic results theinventors observed (FIG. 9). Treatment with GrB/4D5/26 markedlyinhibited phosphorylation of Her2/neu and its downstream molecules Akt,mTOR and ERK, which are critical events in Her2/neu signaling cascade.In contrast, GrB/4D5 showed a comparatively reduced effect on thesepathways. The inventors observed a reduced ER level among all cells.Evidence from other researchers has demonstrated that upregulation ofthe ER pathway in ER- and Her2/neu-positive cell lines with Lapatinibcreates an escape/survival pathway (Wang et al., 2011; Liu et al.,2009), but GrB/4D5/26 appear to be able to inactivate all the signalingpathways in these cells. The inventors also observed the delayingsignaling effects of GrB/4D5/26 on LR cells compared with parental or HRcells, which was in agreement with the apoptotic cell death resultsobserved for the LR cells. Notably, there was an increased mRNA andprotein level of PI-9 in this resistant line but not in the parental orHR cells (FIGS. 15 and 16). Taken together, these results suggest thatactivation of the ER pathway upregulates the expression of PI-9, whichresults in a slight inhibition of GrB/4D5/26 activity and a delay inapoptotic cell death compared to parental cells.

The inventor's investigation suggests that the GrB/4D5/26 fusion is morecytotoxic than GrB/4D5 construct to Her2/neu-positive cells, even thosethat have acquired resistance to the traditional Her2/neu therapeuticagents Herceptin and Lapatinib. The cytotoxicity results coincide withthe observed effects on signal transduction and monitoring thesepathways may be useful as a monitor of drug efficacy.

Effects of GrB/4D5/26 on the MDR-1 Expressing Cells.

Multidrug resistance (MDR) is a phenomenon that results from variousreasons. The most-characterized cause of MDR is the overexpression of a170-kDa membrane glycoprotein known as P-glycoprotein (Pgp). To verifythe effects of GrB-based fusions on the Her2/neu positive cells withMDR-1 expression, the inventors generated the BT474 M1 MDR-1 cells bythe transfection of plasmid pHaMDR1 to parental BT474 M1 cells. As Table13 shown, compared with parental cells, BT474 M1 MDR-1 showed 209-foldresistance to Taxol, and 89-fold resistance to Vinblastin. However, theinventors could not observe the cross-resistance of MDR-1 cells toGrB/4D5 and GrB/4D5/26 constructs. Therefore, GrB-based fusionconstructs demonstrate a wide range cytotoxicity to target cells eventhose with acquired resistance to chemotherapeutic agents.

TABLE 13 Cytotoxicity of Chemical agents and GrB-based fusions on MDR-1expressing cells. IC50 (nM) BT474 M1 BT474 M1 MDR-1 Fold Resistance*Taxol 5.2 1047.3 209 Vinblastin 1.3 105.1 89 GrB/4D5 311.8 318.9 1GrB/4D5/26 34.1 35.5 1 *Fold Resistance (F.R.) represents IC₅₀ of agenton BT474 M1 MDR-1 cells/that on BT474 M1 parental cells.

Antitumor Activity of GrB/4D5/26 Fusions in Xenograft Models.

The inventors used BALB/c nude mice to evaluate the in vivo effect ofGrB/4D5/26 against aggressive breast cancer after systemicadministration. Each mouse received a weekly subcutaneous injection of 3mg/kg estradiol cypionate (Jerome et al., 2006; Gully et al., 2010)starting 2 weeks prior to the injection of 1×10⁷ BT474 M1 cells into theright flank. On the third day after cell inoculation, mice were injectedintravenously (tail vein) either with saline or GrB/4D5/26 (44 mg/kg)five times per week for 2 weeks. Animals were monitored, and tumors weremeasured (calipers) for an additional 50 days. Compared with saline,GrB/4D5/26 greatly slowed tumor progression over 50 days of observation(FIG. 10A). There were no obvious toxic effects of GrB/4D5/26 on mice atthis dose suggesting that the maximum tolerated dose at this schedulehad not been reached.

Finally, the inventors determined the localization of GrB/4D5/26 afteradministration to mice bearing BT474 M1 tumors. Twenty-four hours afterthe final injection of saline or GrB/4D5/26, the mice were sacrificedand tumor samples were frozen immediately in preparation for sectionslides. The sample slides were incubated with either anti-GrB antibody(FITC-conjugated secondary antibody) or a terminal deoxynucleotidyltransferase-mediated nick end labeling (TUNEL) reaction mixture, as wellas with an anti-mouse CD31 antibody (phycoerythrin-conjugated secondaryantibody), and were further subjected to nuclear counterstaining withHoechst 33342. Immunofluorescence observation was performed under aZeiss Axioplan 2 imaging microscope (Carl Zeiss).

Immunofluorescence staining confirmed that GrB/4D5/26 localized quicklyand specifically in tumor tissue (FIG. 10B). This observation furthersuggested that GrB/4D5/26 can effectively target tumor cellsoverexpressing Her2/neu in vivo and can demonstrate significant tumorgrowth-suppressive effects in the absence of observable toxicity.Staining of tumor tissue nuclei with TUNEL (FIG. 10C) clearlydemonstrated that the tumor tissues displayed apoptotic nuclei in theGrB/4D5/26 treatment group. In addition, the intratumoral distributionof GrB/4D5/26 appeared to concentrate primarily in areas with extensiveapoptotic response (compare Grb/4D5/26 distribution in FIG. 10B, withTUNEL staining in FIG. 10C).

In these studies, the inventors constructed novel human anti-Her2/neuimmunotoxins containing human GrB as an apoptosis-inducing effector. GrBappears to be an ideal payload for targeted therapeutic applications inpart because this serine protease exerts a multi-modal and well-knownmechanism of cytotoxic action (Trapani and Sutton, 2003; Chowdhurty andLieberman, 2008). Of interest, this study found that inhibitors ofcaspase activation had little impact on the overall cytotoxicity of theconstruct attesting to the presence of multiple, redundant,pro-apoptotic pathways activated by this molecule and suggesting thatemergence of resistance to this class of agents may be difficult from abiological perspective.

In a nominal cytotoxic process, GrB penetrates directly into targetcells through the action of perforin-mediated transmembrane pores. Thisprocess bypasses the lysosomal compartment allowing GrB accessibilitydirectly to cytosolic substrates (Motyka et al., 2000). Internalizationof GrB through antibody-mediated events provides tumor cell specificitybut in the case of Her2/neu, internalization likely proceeds through thelysosomal compartment. For the inventor's optimal construct, theinventors included a 26-residue, fusogenic peptide. At neutral pH, thispeptide has a random configuration, but under acidic lysosomalconditions, this peptide assumes an amphipathic helix thereby disruptingthe lysosomal membrane allowing improved delivery of the fusionconstruct into the cytosol (Turk et al., 2002).

Dalken et al. (2006) described the construction and biological activityof Her2/neu targeted fusion construct GrB/FRP5. This agent was shown tobe specifically cytotoxic to target cells with IC₅₀ values in thesubnanomolar range but the cytotoxic activity was dependent on theaddition of the lysomotropic agent chloroquine. In the absence ofchloroquine, the cytotoxicity of the agent was reduced 10-300 fold thussuggesting that the construct may have been primarily sequestered intothe lysozomal compartment and not available to activate apoptoticcascade mechanisms. The incorporation of the fusogenic, pH-sensitivepeptide 26 in the inventor's construct appeared to circumvent the needfor a lysomotropic agent to augment the activity of GrB fusion and itprovided a greater concentration of target protein in the cell. The useof this peptide did not appear to impact the enzymatic activity of theGrB component nor did it influence the binding activity of the 4D5 toHer2/neu receptor. Finally, the presence of the 26 component did notappear to augment the nonspecific toxicity of the construct againstantigen-negative cells in vitro nor did it increase the apparenttoxicity of the construct during i.v. administration in the inventor'sxenograft studies.

The antitumor efficacy studies demonstrated that GrB/4D5/26 in the BT474M1 xenograft model was effective at a total dose of 44 mg/kg. This dosetranslates to a total dose of ˜140 mg/m². Clinical dose levels of theT-DM1 conjugate are approximately 3.6 mg/kg (˜280 mg/m²), which isapproximately 2 fold higher than the inventor's extrapolated clinicaldoses for the GrB construct. The inventor's study demonstrated thatthere were no deaths or weight loss during the treatment schedulesuggesting the safety and tolerability of this agent. Although theinventors did not observe complete regression of tumor xenografts,alternative schedules or higher doses need to be employed.

The Her2/neu-targeted therapeutic agents Herceptin and Lapatinib havesignificantly improved outcomes in cancer treatment, but their use islimited by resistance and tolerability issues (Garrett and Arteaga,2011; Bedard et al., 2009). Evaluating the cytotoxicity offunctionalized GrB fusions to HR or LR cells represents an importantstep. The inventor's results suggested that GrB/4D5/26 inhibits theproliferation and survival of resistant cells as a result ofcaspase-dependent and independent apoptotic effects. In addition, theinventor's investigation into cellular signaling indicated thatGrB/4D5/26 could efficiently downregulate the phosphorylation ofHer2/neu and ER family members, resulting in inhibition of both PI3K/Aktand Ras/ERK pathways.

The development of multidrug resistance mechanisms affecting groups oftherapeutic agents has been shown to be a central problem resulting inreduced response in cancer treatment (Szakacs et al., 2006; Hilgeroth etal., 2012). The emergence of MDR phenotypes could also be a seriousproblem for the application of ADCs (Hurvitz and Kakkar, 2012; Murphyand Morris, 2012). Studies by Kovtun et al. (2010) reported that ADCsutilizing PEG-based hydrophilic linkers showed higher retention in MDR-1expressing cells than similar conjugates made with the nonpolar linkerSMCC which is found in T-DM1. Therefore, the emergence of MDR mayprovide cross-resistance to T-DM1, due to the efflux of free drug uponintracellular release from the antibody. In contrast, the currentstudies demonstrate that expression of MDR does not providecross-resistance to GrB-based fusion constructs and this appears to be asignificant advantage over the conventional ADC approach.

The only intracellular inhibitor of human GrB is the nucleocytoplasmicserpin, PI-9. PI-9 has been found to be endogenously expressed inlymphocytes, dendritic cells and mast cells, for self-protection againstGrB-mediated apoptosis (Trapani and Sutton, 2003; Chowdhury andLieberman, 2008). This may suggest that the endogenous PI-9 level incancer cells could inhibit the GrB activity of the inventor's targetmolecules. However, the inventor's studies did not show any relationshipbetween PI-9 levels and cell sensitivity to GrB/4D5/26 in Her2/neupositive cells.

The inventors examined GrB sensitivity against Lapatinib-resistant cellsand found these cells showed a slight (2-fold) increase in theGrB/4D5/26 IC₅₀. This coincided with an upregulation of PI-9 leading toa delay in apoptosis. This upregulation may be the indirect result of ERpathway changes induced by Lapatinib resistance. Therefore, in the celllines that are both ER- and Her2-positive, for which upregulation of theER pathway may occur as an escape pathway, the endogenous GrB inhibitorPI-9 could be upregulated to inhibit GrB activity.

In conclusion, the foregoing studies demonstrate that a novel Her2/neutargeted functionalized GrB fusion constructs employing the pH-sensitivefusogenic peptide 26 as an endosomolytic domain efficiently promotes therelease of GrB into the cytoplasm, resulting in apoptotic cell death inHer2/neu-positive cancer cells. This fusogenic peptide could be usefulfor studying GrB-induced apoptosis without the requirement of perforinor chloroquine. In addition, the studies demonstrate that tumor cellshighly resistant to either Lapatinib or trastuzumab (Herceptin®) and thecells with MDR-1 expression resistant to chemotherapeutic agents werenot cross-resistant to the GrB-based fusion protein. Although theinduction of PI-9 expression in LR cells delayed the apoptoticcytotoxicity of GrB/4D5/26, this agent had an IC₅₀ value that was only2-fold higher than parental cells, despite the fact that resistant cellswere more than 200-fold resistant to Lapatinib.

Example 12—Construction of Cleavable Carboxyl Terminal GrB Fusions

GrB fusion constructs were constructed comprising a targetingpolypeptide positioned at the N-terminus relative to the GrB codingsequence. The resulting fusion proteins are engineered to include aprotease cleavage site that, after protease cleavage releases an activeGrB enzyme (i.e., have a free isoleucine at the amino terminus).

Initial constructs tested comprised a targeting moiety (e.g., anantibody)+caspase cleavable peptide+Granzyme B (“the insert”). A linker,such as the G₄S linker or a 218 linker, may also be incorporated betweenthe targeting moiety and the cleavable peptide. One caspase cleavablepeptide sequence of particular interest is the YVDEVD↓ (SEQ ID NO: 25;which can be followed by the GrB amino acid sequence), where the “↓”indicates the cleavage site. In some aspects, the caspase-3 cleavablepeptide may be substituted with a peptide cleavable by a differentprotease, such as another caspase or furin.

For the initial test constructs the anti-Her2/neu scFv 4D5 sequence wasgrafted onto a human IgG1 framework to generate the “4D5-IgG1” baseconstruct. This grafted antibody was subject to testing of affinity tothe Her2 ECD as compared to Herceptin®. These studies, shown in FIG. 17,confirm that the two antibodies display similar target affinity. Usingthe 4D5-IgG1 base, several GrB fusion proteins were generated and testedfor cytotoxic activity against appropriate cell lines. The constructsproduced were as follows:

4D5-Ac—

The 4D5-IgG1 heavy chain was fused to the N-terminus of GrB, such thatthe 4D5 heavy chain and GrB were separated by the caspase cleavablelinker detailed above. In this case the GrB coding sequence comprisesthe N51S and C210A point mutations and included the INF7 translocationpeptide at the C-terminus. Thus, from N- to C-terminus the heavy chainof the construct comprises 4D5IgG1 heavy chain—caspase cleavablelinker—GrB—INF7 (see, e.g., FIG. 18, lower left panel).

4D5-AfNI—

The 4D5-IgG1 heavy chain was fused to the N-terminus of GrB, such thatthe 4D5 heavy chain and GrB were separated by the furin cleavablelinker. The GrB coding sequence comprises the N51S and C210A pointmutations. Thus, from N- to C-terminus the heavy chain of the constructcomprises 4D5IgG1 heavy chain—furin cleavable linker—GrB (see, e.g.,FIG. 18, lower left panel).

4D5-BfNI—

The 4D5-IgG1 light chain was fused to the N-terminus of GrB, such thatthe 4D5 light chain and GrB were separated by a furin cleavable linker.The GrB coding sequence comprises the N51S and C210A point mutations.Thus, from N- to C-terminus the light chain of the construct comprises4D5IgG1 light chain—furin cleavable linker—GrB (see, e.g., FIG. 18,upper right panel).

4D5-AeafNI—

The 4D5-IgG1 heavy chain was fused to the N-terminus of GrB, such thatthe 4D5 heavy chain and GrB were separated by the caspase cleavablelinker detailed above. In this case the GrB coding sequence comprisesthe N51S and C210A point mutations in addition to K27E and R28A. Thus,from N- to C-terminus the heavy chain of the construct comprises 4D5IgG1heavy chain—caspase cleavable linker—GrB (see, e.g., FIG. 18, lower leftpanel).

IgG-Ac—

A murine anti-Her2 IgG1 heavy chain was fused to the N-terminus of GrB,such that the 4D5 heavy chain and GrB were separated by the caspasecleavable linker detailed above. In this case the GrB coding sequencecomprises the N51S and C210A point mutations and included the INF7translocation peptide at the C-terminus. Thus, from N- to C-terminus theheavy chain of the construct comprises a murine IgG1 heavy chain—caspasecleavable linker—GrB—INF7 (see, e.g., FIG. 18, lower left panel).

The constructs above were expressed in mammalian cells from abicistronic expression vector arranged such that the heavy and lightchain antibody polypeptides (or fusions thereof) were secreted.Assembled antibody fusion constructs were purified from the cell media.These constructs were then tested for cytotoxic activity relative toHer2-expressing SKBR3 cells or control MCF-7 cells (that do not expressHer2). The results of these studies (shown below in Table 14)demonstrate that the GrB fusion antibodies all showed robust cytotoxicactivity with at least 4× lower IC₅₀ as compared to Herceptin®.

TABLE 14 Cytotoxicity of GrB/IgG constructs 4D5- 4D5- Cell 4D5-Ac AfNI4D5-BfNI AeafNI IgG-Ac Herceptin ® line IC₅₀ (nm) SKBR3 68 98 108 73 152454 MCF-7 >200 >200 >200 >200 >200 >1000

Additional studies were undertaken with GrB fusions to HMEL scFv. Forthese studies the cytotoxicity of constructs “HCB” (HMEL scFv-G₄S-YVDEVD(SEQ ID NO: 25)-GrB) was compared to control constructs “WH”(GrB-G₄S-INF7-HMEL scFv) and “HNB” (HMEL scFv-G₄S-GrB) were compared onAAB527 versus MEF3.5−/− cells (see FIG. 19, lower panel for constructschematics). The results of these studies shown in FIG. 19 demonstratethat, as expected, none of the constructs had significant activityrelative to cells lacking the target receptor (right panel graph). Incontrast only the WH and HCB constructs had significant activityrelative to the AAB527 cells, showing the specific cleavage of the GrBinto an active form was required for cytotoxic activity. Further studieswill be undertaken to test constructs that comprise both a heavy chainand light chain GrB fusion.

Example 13—GrB Fusion Constructs Comprising scFv Regions Fused to Fc

Further GrB fusion constructs were designed and constructed thatincluded scFv regions as well as antibody Fc domains. For theseconstructs GrB may be fused to be positioned N-terminal relative to theantibody sequences or at the c-terminus (via a cleavable linker asdetailed above). Thus constructs can comprise the general structureGrB-Fc-scFv (see, e.g., FIG. 18, lower right panel) or scFv-Fc-cleavablelinker-GrB.

As an initial test of this arrangement a construct was producedcomprising GrB-Fc-IT4 (scFv). Specifically, the IT4 scFv targets theproduct of the tumor necrosis factor receptor superfamily, member 12A(TNFRSF12A) gene and was previously described in Zhou et al., 2011,which is incorporated herein by reference. The sequence of the fusionprotein produced by the construct is provided as SEQ ID NO: 45. Theconstructs was expressed and purified as detailed supra and tested foractivity against a panel of cells lines (using GrB alone a control).Results shown below in Table 15 demonstrate that the constructs werehighly active with IC₅₀ values measure as low as 3.

TABLE 15 Cytotoxicity of GrB-Fc-IT4 constructs IC50 (nM) Cell linesGrB-Fc-IT4 GrB A549 19 602 AsPc-1 17 1297 Capan-2 21 >3200 Capan-1 372344 L3.6p1 35 1215 H358 121 >3200 H520 62 259 H1437 14 >3200H1975 >114 >3200 H2073 >114 >3200 H3255 27 2359 HCC827 114 1406 HCC227919 526 MDA-MB-435 12 445 WM35P2N1 144 1543 WM35 3 >1923 SB2 17 >1923A375 73 >1923 SK-MEL-28 >284 >1923 MCF-7 35 >1923 MDA-MB-231 17 >1931MDA-MD-231- 3 3554 Luc HT-29 118 >3200 MEF 3.5 −/− >114 >3200

Example 14—Studies with Additional GrB-VEGF Fusion Constructs

Additional studies were undertaken to study the serum stability andcytotoxicity of various GrB mutants fused to a VEGF targeting moiety.The constructs tested were as follows: GrB/VEGF₁₂₁; EA-GrB/VEGF₁₂₁ (GrBmutant with the K27E, R28A point mutations); LA-GrB/VEGF₁₂₁ (GrB mutantwith the K27L, R28A point mutations); EAPVPN-GrB/VEGF₁₂₁ (⁸²PKN⁸⁴ loopof wt GrB was mutated to PVPN and comprising the K27E, R28A pointmutations); PVPN-GrB/VEGF₁₂₁ (⁸²PKN⁸⁴ loop of wt GrB was mutated toPVPN); LP-GrB/VEGF₁₂₁ (addition of His-tag, thrombin cleavage site andCaspase-3 cleavage site (DEVD) immediately upstream of the GrBN-terminus). These constructs were tested for enzymatic activityfollowing incubation in serum (FBS) for 4 hours or incubation in PBS for4 hours. The results of these studies are shown in Table 16 below. Thesestudies showed that both the “EA” and “LA” mutations were able to remainsignificantly more active than control constructs following serumincubation.

The same constructs were tested for cytotoxic activity followingincubation for 4 hours in serum (FBS) or in PBS. The results of thesestudies are shown in Table 17. These data demonstrate that, even afterserum incubation, the “EA” and “LA” mutant targeting constructs remainhighly active and specific against target cells. Crucially, however, theLP-GrB/VEGF₁₂₁ construct also remained highly active even after exposureto serum. It is hypothesized that the construct is protected by virtueof the fact that it is inactive until cellular uptake upon, whichcaspase cleavage activated GrB enzymatic activity.

TABLE 16 Resistance of GrB mutants to inactivation by serum EnzymaticActivity remaining (%) Construct PBS (4 h) FBS (4 h) GrB/VEGF₁₂₁  82 36EA-GrB/VEGF₁₂₁  79 76 LA-GrB/VEGF₁₂₁ 100 51 EAPVPN-GrB/VEGF₁₂₁ ND NDPVPN-GrB/VEGF₁₂₁ 100 34 LP-GrB/VEGF₁₂₁ Inactive Inactive

TABLE 17 Cytotoxicity of GrB mutants with or without pre-incubation inserum IC₅₀ (nM) on IC₅₀ (nM) on PEA/VEGFR-2 cells PEA/VEGFR-1 cellsConstruct PBS (4 h) FBS (4 h) PBS (4 h) FBS (4 h) GrB/VEGF₁₂₁4 >100  >100 >100 EA-GrB/VEGF₁₂₁ 8 39 >100 >100 LA-GrB/VEGF₁₂₁ 863 >100 >100 EAPVPN-GrB/VEGF₁₂₁ 5 Not tested >100 Not testedPVPN-GrB/VEGF₁₂₁ 8 Not tested >100 Not tested LP-GrB/VEGF₁₂₁ 2 67 >100>100

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed is:
 1. A recombinant polypeptide comprising a Granzyme B(GrB) coding sequence at least 85% identical to SEQ ID NO: 1 wherein theGrB coding sequence comprises an amino acid substitution or deletion atthe position corresponding to Cys 210, wherein the polypeptide isconjugated to or fused with a cell binding moiety.
 2. The polypeptide ofclaim 1, wherein the polypeptide further comprises an amino acidsequence comprising a Cys, wherein the amino acid sequence is positionedC-terminally relative to the GrB coding sequence.
 3. The polypeptide ofclaim 2, wherein the polypeptide further comprises the sequence SSCSGSA(SEQ ID NO: 12) positioned C-terminally relative to the GrB codingsequence.
 4. The polypeptide of claim 1, wherein the polypeptidecomprises an Ala substitution at the position corresponding to Cys 210.5. The polypeptide of claim 1, wherein the polypeptide is conjugated tothe cell binding moiety by a thioester linkage.
 6. The polypeptide ofclaim 1, wherein the polypeptide fused with a cell binding moietypositioned C-terminally relative to the GrB coding sequence.
 7. Thepolypeptide of claim 1, wherein the cell binding moiety is a VEGF, BLyS,an antibody or a cell-binding portion of any of the foregoing.
 8. Thepolypeptide of claim 7, wherein the antibody is a monoclonal, chimericantibody, Fab′, Fab, F(ab′)2, single domain antibody, Fv, or singlechain Fv (scFv) antibody.
 9. The polypeptide of claim 7, wherein theantibody is a human antibody, a humanized antibody or a deimmunizedantibody.
 10. The polypeptide of claim 7, wherein the antibody is a15A8, ZME-018, ScFvMEL, cetuximab or trastuzumab antibody.
 11. Thepolypeptide of claim 1, wherein the cell binding moiety binds to aprotein, carbohydrate or lipid expressed on cancer cells.
 12. Thepolypeptide of claim 1, wherein the cell binding moiety binds to GP240,5T4, HER1, HER2, CD-33, CD-38, fltI, Flk-1, CEA, FGFR3, IGFBP2 orIGF-1R.
 13. The polypeptide of claim 1, wherein the polypeptide or cellbinding moiety is further conjugated to an imaging agent.
 14. Thepolypeptide of claim 1, wherein the Granzyme B (GrB) coding sequence isat least 90% identical to SEQ ID NO:
 1. 15. The polypeptide of claim 14,wherein the Granzyme B (GrB) coding sequence is at least 95% identicalto SEQ ID NO:
 1. 16. The polypeptide of claim 15, wherein the Granzyme B(GrB) coding sequence is identical to SEQ ID NO: 1.